Compounds and methods for treating mitochondria-associated diseases

ABSTRACT

Compounds, compositions and methods are disclosed for treating mitochondria-associated diseases, such as cancer, psoriasis, stroke, Alzheimer&#39;s Disease and diabetes. The compounds of this invention have structure (I) below, including stereoisomers, prodrugs and pharmaceutically acceptable salts thereof, wherein Ar and L are as defined herein. The methods of this invention are directed to treating a mitochondria-associated disease by administering to a warm-blooded animal in need thereof an effective amount of a compound of structure (I), typically in the form of a pharmaceutical composition.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/082,998 filed Apr. 24, 1998.

TECHNICAL FIELD

[0002] The present invention relates generally to compounds and methodsfor treating mitochondria-associated diseases and, more particularly, to(i) diseases and disorders in which free radical mediated oxidativeinjury leads to tissue degeneration, (ii) diseases and disorders inwhich cells inappropriately undergo programmed cell death (apoptosis),leading to tissue degeneration, or (iii) diseases and disorders, such ascancer, in which some cells in the body fail to undergo apoptosis withdetrimental consequences to the body as a whole. More specifically, thepresent invention relates to compositions and methods for treating suchdisease and disorders through the use of compounds which function as,respectively, (1) mitochondria protecting agents, (2) anti-apoptoticagents, or (3) pro-apoptotic agents.

BACKGROUND OF THE INVENTION

[0003] Mitochondria are the main energy source in cells of higherorganisms, and these organelles provide direct and indirect biochemicalregulation of a wide array of cellular respiratory, oxidative andmetabolic processes (for a review, see Ernster and Schatz, J. Cell Biol.91:227s-255s, 1981). These include electron transport chain (ETC)activity, which drives oxidative phosphorylation to produce metabolicenergy in the form of adenosine triphosphate (ATP), and which alsounderlies a central mitochondrial role in intracellular calciumhomeostasis. In addition to their role in metabolic processes,mitochondria are also involved in the genetically programmed cellsuicide sequence known as “apoptosis” (Green and Reed, Science281:1309-1312, 1998; Susin et al., Biochim. et Biophys. Acta1366:151-165, 1998).

[0004] Defective mitochondrial activity, including but not limited tofailure at any step of the elaborate multi-complex mitochondrialassembly, known as the electron transport chain (ETC), may result in (i)decreases in ATP production, (ii) increases in the generation of highlyreactive free radicals (e.g., superoxide, peroxynitrite and hydroxylradicals, and hydrogen peroxide), (iii) disturbances in intracellularcalcium homeostasis and (iv) the release of factors (such as such ascytochrome c and “apoptosis inducing factor”) that initiate or stimulatethe apoptosis cascade. Because of these biochemical changes,mitochondrial dysfunction has the potential to cause widespread damageto cells and tissues.

[0005] A number of diseases and disorders are thought to be caused by orbe associated with alterations in mitochondrial metabolism and/orinappropriate induction or suppression of mitochondria-related functionsleading to apoptosis. These include, by way of example and notlimitation, chronic neurodegenerative disorders such as Alzheimer'sdisease (AD) and Parkinson's disease (PD); auto-immune diseases;diabetes mellitus, including Type I and Type II; mitochondria associateddiseases, including but not limited to congenital muscular dystrophywith mitochondrial structural abnormalities, fatal infantile myopathywith severe mtDNA depletion and benign “later-onset” myopathy withmoderate reduction in mtDNA, MELAS (mitochondrial encephalopathy, lacticacidosis, and stroke) and MIDD (mitochondrial diabetes and deafness);MERFF (myoclonic epilepsy ragged red fiber syndrome); arthritis; NARP(Neuropathy; Ataxia; Retinitis Pigmentosa); MNGIE (Myopathy and externalophthalmoplegia; Neuropathy; Gastro-Intestinal; Encephalopathy), LHON(Leber's; Hereditary; Optic; Neuropathy), Kearns-Sayre disease;Pearson's Syndrome; PEO (Progressive External Ophthalmoplegia); Wolframsyndrome DIDMOAD (Diabetes Insipidus, Diabetes Mellitus, Optic Atrophy,Deafness); Leigh's Syndrome; dystonia; schizophrenia; andhyperproliferative disorders, such as cancer, tumors and psoriasis.

[0006] According to generally accepted theories of mitochondrialfunction, proper ETC respiratory activity requires maintenance of anelectrochemical potential (ATm) in the inner mitochondrial membrane by acoupled chemiosmotic mechanism. Conditions that dissipate or collapsethis membrane potential, including but not limited to failure at anystep of the ETC, may thus prevent ATP biosynthesis and hinder or haltthe production of a vital biochemical energy source. Altered ordefective mitochondrial activity may also result in a catastrophicmitochondrial collapse that has been termed “mitochondrial permeabilitytransition” (MPT). In addition, mitochondrial proteins such ascytochrome c and “apoptosis inducing factor” may dissociate or bereleased from mitochondria due to MPT (or the action of mitochondrialproteins such as Bax), and may induce proteases known as caspases and/orstimulate other events in apoptosis (Murphy, Drug Dev. Res. 46:18-25,1999).

[0007] Defective mitochondrial activity may alternatively oradditionally result in the generation of highly reactive free radicalsthat have the potential of damaging cells and tissues. These freeradicals may include reactive oxygen species (ROS) such as superoxide,peroxynitrite and hydroxyl radicals, and potentially other reactivespecies that may be toxic to cells. For example, oxygen free radicalinduced lipid peroxidation is a well established pathogenetic mechanismin central nervous system (CNS) injury such as that found in a number ofdegenerative diseases, and in ischemia (i.e., stroke). (Mitochondrialparticipation in the apoptotic cascade is believed to also be a keyevent in the pathogenesis of neuronal death.)

[0008] There are, moreover, at least two deleterious consequences ofexposure to reactive free radicals arising from mitochondrialdysfunction that adversely impact the mitochondria themselves. First,free radical mediated damage may inactivate one or more of the myriadproteins of the ETC. Second, free radical mediated damage may result incatastrophic mitochondrial collapse that has been termed “transitionpermeability”. According to generally accepted theories of mitochondrialfunction, proper ETC respiratory activity requires maintenance of anelectrochemical potential in the inner mitochondrial membrane by acoupled chemiosmotic mechanism. Free radical oxidative activity maydissipate this membrane potential, thereby preventing ATP biosynthesisand/or triggering mitochondrial events in the apoptotic cascade.Therefore, by modulating these and other effects of free radicaloxidation on mitochondrial structure and function, the present inventionprovides compositions and methods for protecting mitochondria that arenot provided by the mere determination of free radical induced lipidperoxidation.

[0009] For example, rapid mitochondrial permeability transition likelyentails changes in the inner mitochondrial transmembrane proteinadenylate translocase that results in the formation of a “pore”. Whetherthis pore is a distinct conduit or simply a widespread leakiness in themembrane is unresolved. In any event, because permeability transition ispotentiated by free radical exposure, it may be more likely to occur inthe mitochondria of cells from patients having mitochondria associateddiseases that are chronically exposed to such reactive free radicals.

[0010] Altered mitochondrial function characteristic of the mitochondriaassociated diseases may also be related to loss of mitochondrialmembrane electrochemical potential by mechanisms other than free radicaloxidation, and such transition permeability may result from direct orindirect effects of mitochondrial genes, gene products or relateddownstream mediator molecules and/or extramitochondrial genes, geneproducts or related downstream mediators, or from other known or unknowncauses. Loss of mitochondrial potential therefore may be a criticalevent in the progression of mitochondria associated or degenerativediseases.

[0011] Diabetes mellitus is a common, degenerative disease affecting 5to 10 percent of the population in developed countries. The propensityfor developing diabetes mellitus is reportedly maternally inherited,suggesting a mitochondrial genetic involvement. (Alcolado, J. C. andAlcolado, R., Br. Med. J 302:1178-1180 (1991); Reny, S. L.,International J. Epidem. 23:886-890 (1994)). Diabetes is a heterogenousdisorder with a strong genetic component; monozygotic twins are highlyconcordant and there is a high incidence of the disease among firstdegree relatives of affected individuals.

[0012] At the cellular level, the degenerative phenotype that may becharacteristic of late onset diabetes mellitus includes indicators ofaltered mitochondrial respiratory function, for example impaired insulinsecretion, decreased ATP synthesis and increased levels of reactiveoxygen species. Studies have shown that diabetes mellitus may bepreceded by or associated with certain related disorders. For example,it is estimated that forty million individuals in the U.S. suffer fromlate onset impaired glucose tolerance (IGT). IGT patients fail torespond to glucose with increased insulin secretion. A small percentageof IGT individuals (5-10%) progress to insulin deficient non-insulindependent diabetes (NIDDM) each year. Some of these individuals furtherprogress to insulin dependent diabetes mellitus (IDDM). These forms ofdiabetes mellitus, NIDDM and IDDM, are associated with decreased releaseof insulin by pancreatic beta cells and/or a decreased end-organresponse to insulin. Other symptoms of diabetes mellitus and conditionsthat precede or are associated with diabetes mellitus include obesity,vascular pathologies, peripheral and sensory neuropathies, blindness anddeafness.

[0013] Due to the strong genetic component of diabetes mellitus, thenuclear genome has been the main focus of the search for causativegenetic mutations. However, despite intense effort, nuclear genes thatsegregate with diabetes mellitus are known only for rare mutations inthe insulin gene, the insulin receptor gene, the adenosine deaminasegene and the glucokinase gene. Accordingly, mitochondrial defects, whichmay include but need not be limited to defects related to the discretenon-nuclear mitochondrial genome that resides in mitochondrial DNA, maycontribute significantly to the pathogenesis of diabetes mellitus(Anderson, Drug Dev. Res. 46:67-79, 1999).

[0014] Parkinson's disease (PD) is a progressive, chronic, mitochondriaassociated neurodegenerative disorder characterized by the loss and/oratrophy of dopamine-containing neurons in the pars compacta of thesubstantia nigra of the brain. Like Alzheimer's Disease (AD). PD alsoafflicts the elderly. It is characterized by bradykinesia (slowmovement), rigidity and a resting tremor. Although L-Dopa treatmentreduces tremors in most patients for a while, ultimately the tremorsbecome more and more uncontrollable, making it difficult or impossiblefor patients to even feed themselves or meet their own basic hygieneneeds.

[0015] It has been shown that the neurotoxin1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces parkinsonismin animals and man at least in part through its effects on mitochondria.MPTP is converted to its active metabolite, MPP+, in dopamine neurons;it then becomes concentrated in the mitochondria. The MPP+thenselectively inhibits the mitochondrial enzyme NADH:ubiquinoneoxidoreductase (“Complex I”), leading to the increased production offree radicals, reduced production of adenosine triphosphate, andultimately, the death of affected dopamine neurons.

[0016] Mitochondrial Complex I is composed of 40-50 subunits; most areencoded by the nuclear genome and seven by the mitochondrial genome.Since parkinsonism may be induced by exposure to mitochondrial toxinsthat affect Complex I activity, it appears likely that defects inComplex I proteins may contribute to the pathogenesis of PD by causing asimilar biochemical deficiency in Complex I activity. Indeed, defects inmitochondrial Complex I activity have been reported in the blood andbrain of PD patients (Parker et al., Am. J. Neurol. 26:719-723, 1989;Swerdlow and Parker, Drug Dev. Res. 46:44-50, 1999).

[0017] Similar theories have been advanced for analogous relationshipsbetween mitochondrial defects and other neurological diseases, includingAlzheimer's disease, Leber's hereditary optic neuropathy, schizophrenia,“mitochondrial encephalopathy, lactic acidosis, and stroke” (MELAS), and“myoclonic epilepsy ragged red fiber syndrome” (MERRF).

[0018] For example, Alzheimer's disease (AD) is a chronic, progressiveneurodegenerative disorder that is characterized by loss and/or atrophyof neurons in discrete regions of the brain, and that is accompanied byextracellular deposits of β-amyloid and the intracellular accumulationof neurofibrillary tangles. It is a uniquely human disease, affectingover 13 million people worldwide. It is also a uniquely tragic disease.Many individuals who have lived normal, productive lives are slowlystricken with AD as they grow older, and the disease gradually robs themof their memory and other mental faculties. Eventually, they cease torecognize family and loved ones, and they often require continuous careuntil their eventual death.

[0019] There is evidence that defects in oxidative phosphorylationwithin the mitochondria are at least a partial cause of sporadic AD. Theenzyme cytochrome c oxidase (COX), which makes up part of themitochondrial electron transport chain (ETC), is present in normalamounts in AD patients; however, the catalytic activity of this enzymein AD patients and in the brains of AD patients at autopsy has beenfound to be abnormally low. This suggests that the COX in AD patients isdefective, leading to decreased catalytic activity that in some fashioncauses or contributes to the symptoms that are characteristic of AD.

[0020] One hallmark pathology of AD is the death of selected neuronalpopulations in discrete regions of the brain. Cell death in AD ispresumed to be apoptotic because signs of programmed cell death (PCD)are seen and indicators of active gliosis and necrosis are not found(Smale et al., Exp. Neurolog. 133:225-230, 1995; Cotman et al., Molec.Neurobiol. 10:19-45, 1995.) The consequences of cell death in AD,neuronal and synaptic loss, are closely associated with the clinicaldiagnosis of AD and are highly correlated with the degree of dementia inAD (DeKosky et al., Ann. Neurology 2757-464, 1990).

[0021] Mitochondrial dysfunction is thought to be critical in thecascade of events leading to apoptosis in various cell types (Kroemer etal., FASEB J 9:1277-1287, 1995), and may be a cause of apoptotic celldeath in neurons of the AD brain. Altered mitochondrial physiology maybe among the earliest events in PCD (Zamzami et al., J. Exp. Med.182:367-77, 1995; Zamzami et al., J. Exp. Med. 181:1661-72, 1995) andelevated reactive oxygen species (ROS) levels that result from suchaltered mitochondrial function may initiate the apoptotic cascade(Ausserer et al., Mol. Cell. Biol. 14:5032-42, 1994). In several celltypes, including neurons, reduction in the mitochondrial membranepotential (ΔΨm) precedes the nuclear DNA degradation that accompaniesapoptosis. In cell-free systems, mitochondrial, but not nuclear,enriched fractions are capable of inducing nuclear apoptosis (Newmeyeret al., Cell 70:353-64, 1994). Perturbation of mitochondrial respiratoryactivity leading to altered cellular metabolic states, such as elevatedintracellular ROS, may occur in mitochondria associated diseases and mayfurther induce pathogenetic events via apoptotic mechanisms.

[0022] Oxidatively stressed mitochondria may release a pre-formedsoluble factor that can induce chromosomal condensation, an eventpreceding apoptosis (Marchetti et al., Cancer Res. 56:2033-38, 1996). Inaddition, members of the Bcl-2 family of anti-apoptosis gene productsare located within the outer mitochondrial membrane (Monaghan et al., J.Histochem. Cytochem. 40:1819-25, 1992) and these proteins appear toprotect membranes from oxidative stress (Korsmeyer et al, Biochim.Biophys. Act. 1271:63, 1995). Localization of Bcl-2 to this membraneappears to be indispensable for modulation of apoptosis (Nguyen et al.,J. Biol. Chem. 269:16521-24, 1994). Thus, changes in mitochondrialphysiology may be important mediators of apoptosis. To the extent thatapoptotic cell death is a prominent feature of neuronal loss in AD,mitochondrial dysfunction may be critical to the progression of thisdisease and may also be a contributing factor in other mitochondriaassociated diseases.

[0023] Focal defects in energy metabolism in the mitochondria, withaccompanying increases in oxidative stress, may be associated with AD.It is well-established that energy metabolism is impaired in AD brain(Palmer et al., Brain Res. 645:338-42, 1994; Pappolla et al., Am. J.Pathol. 140:621-28, 1992; Jeandel et al., Gerontol. 35:275, 1989; Balazset al., Neurochem. Res. 19:1131-37, 1994; Mecocci et al., Ann. Neurol.36:747-751, 1994; Gsell et al., J. Neurochem. 64:1216-23, 1995). Forexample, regionally specific deficits in energy metabolism in AD brainshave been reported in a number of positron emission tomography studies(Kuhl, et al., J. Cereb. Blood Flow Metab. 7:S406, 1987; Grady, et al.,J. Clin. Exp. Neuropsychol. 10:576-96, 1988; Haxby et al., Arch. Neurol.4:753-60, 1990; Azari et al., J. Cereb. Blood Flow Metab. 13:438-47,1993). Metabolic defects in the temporoparietal neocortex of AD patientsapparently presage cognitive decline by several years. Skin fibroblastsfrom AD patients display decreased glucose utilization and increasedoxidation of glucose, leading to the formation of glycosylation endproducts (Yan et al., Proc. Nat. Acad. Sci. U.S.A. 91:7787-91, 1994).Cortical tissue from postmortem AD brain shows decreased activity of themitochondrial enzymes pyruvate dehydrogenase (Sheu et al., Ann. Neurol.17:444-49, 1985) and α-ketoglutarate dehydrogenase (Mastrogiacomo etal., J. Neurochem. 6:2007-2014, 1994), which are both key enzymes inenergy metabolism. Functional magnetic resonance spectroscopy studieshave shown increased levels of inorganic phosphate relative tophosphocreatine in AD brain, suggesting an accumulation of precursorsthat arises from decreased ATP production by mitochondria (Pettegrew etal., Neurobiol. of Aging 15:117-32, 1994; Pettigrew et al., Neurobiol.of Aging 16:973-75, 1995). In addition, the levels of pyruvate, but notof glucose or lactate, are reported to be increased in the cerebrospinalfluid of AD patients, consistent with defects in cerebral mitochondrialelectron transport chain (ETC) activity (Parnetti et al., Neurosci. Lett199:231-33, 1995).

[0024] Signs of oxidative injury are prominent features of AD pathologyand, as noted above, reactive oxygen species (ROS) are criticalmediators of neuronal degeneration. Indeed, studies at autopsy show thatmarkers of protein, DNA and lipid peroxidation are increased in AD brain(Palmer et al., Brain Res. 645:338-42, 1994; Pappolla et al., Am. J.Pathol. 140:621-28, 1992; Jeandel et al., Gerontol. 35:275-82, 1989;Balazs et al., Arch. Neurol. 4:864, 1994; Mecocci et al., Ann. Neurol.36:747-751, 1994; Smith et al., Proc. Nat. Acad. Sci. U.S.A.88:10540-10543, 1991). In hippocampal tissue from AD but not fromcontrols, carbonyl formation indicative of protein oxidation isincreased in neuronal cytoplasm, and nuclei of neurons and glia (Smithet al., Nature 382:120-21, 1996). Neurofibrillary tangles also appear tobe prominent sites of protein oxidation (Schweers et al., Proc. Nat.Acad. Sci. U.S.A. 92:8463, 1995; Blass et al., Arch. Veurol. 4:864,1990). Under stressed and non-stressed conditions incubation of corticaltissue from AD brains taken at autopsy demonstrate increased freeradical production relative to non-AD controls. In addition, theactivities of critical antioxidant enzymes, particularly catalase, arereduced in AD (Gsell et al., J. Neurochem. 64:1216-23, 1995), suggestingthat the AD brain is vulnerable to increased ROS production. Thus,oxidative stress may contribute significantly to the pathology ofmitochondria associated diseases such as AD, where mitochondrialdysfunction and/or elevated ROS may be present.

[0025] Increasing evidence points to the fundamental role ofmitochondrial dysfunction in chronic neurodegenerative diseases (Beal,Biochim. Biophys. Acta 1366: 211-223, 1998), and recent studiesimplicate mitochondria for regulating the events that lead to necroticand apoptotic cell death (Susin et al., Biochim. Biophys. Acta 1366:151-168, 1998). Stressed (by, e.g., free radicals, high intracellularcalcium, loss of ATP, among others) mitochondria may release pre-formedsoluble factors that can initiate apoptosis through an interaction withapoptosomes (Marchetti et al., Cancer Res. 56:2033-38, 1996; Li et al.,Cell 91: 479-89, 1997). Release of preformed soluble factors by stressedmitochondria, like cytochrome c, may occur as a consequence of a numberof events. In any event, it is thought that the magnitude of stress(ROS, intracellular calcium levels, etc.) influences the changes inmitochondrial physiology that ultimately determine whether cell deathoccurs via a necrotic or apoptotic pathway. To the extent that apoptoticcell death is a prominent feature of degenerative diseases,mitochondrial dysfunction may be a critical factor in diseaseprogression.

[0026] In contrast to chronic neurodegenerative diseases, neuronal deathfollowing stroke occurs in an acute manner. A vast amount of literaturenow documents the importance of mitochondrial function in neuronal deathfollowing ischemia/reperfusion injury that accompanies stroke, cardiacarrest and traumatic injury to the brain. Experimental support continuesto accumulate for a central role of defective energy metabolism,alteration in mitochondrial function leading to increased oxygen radicalproduction and impaired intracellular calcium homeostasis, and activemitochondrial participation in the apoptotic cascade in the pathogenesisof acute neurodegeneration.

[0027] A stroke occurs when a region of the brain loses perfusion andneurons die acutely or in a delayed manner as a result of this suddenischemic event. Upon cessation of the blood supply to the brain, tissueATP concentration drops to negligible levels within minutes. At the coreof the infarct, lack of mitochondrial ATP production causes loss ofionic homeostasis, leading to osmotic cell lysis and necrotic death. Anumber of secondary changes can also contribute to cell death followingthe drop in mitochondrial ATP. Cell death in acute neuronal injuryradiates from the center of an infarct where neurons die primarily bynecrosis to the penumbra where neurons undergo apoptosis to theperiphery where the tissue is still undamaged (Martin et al., Brain Res.Bull. 46:281-309, 1998).

[0028] Much of the injury to neurons in the penumbra is caused byexcitotoxicity induced by glutamate released during cell lysis at theinfarct focus, especially when exacerbated by bioenergetic failure ofthe mitochondria from oxygen deprivation (MacManus and Linnik, J.Cerebral Blood Flow Metab. 17:815-832, 1997). The initial trigger inexcitotoxicity is the massive influx of Ca²⁺ primarily through the NMDAreceptors, resulting in increased uptake of Ca²⁺ into the mitochondria(reviewed by Dykens, “Free radicals and mitochondrial dysfunction inexcitotoxicity and neurodegenerative diseases” in Cell Death andDiseases of the Nervous System, V. E. Koliatos and R. R. Ratan, eds.,Humana Press, New Jersey, pages 45-68, 1999). The Ca²⁺ overloadcollapses the mitochondrial membrane potential (ΔΨ_(m)) and inducesincreased production of reactive oxygen species (Dykens, J Neurochem63:584-591, 1994; Dykens, “Mitochondrial radical production andmechanisms of oxidative excitotoxicity” in The Oxygen Paradox, K. J. A.Davies, and F. Ursini, eds., Cleup Press, U. of Padova, pages 453-467,1995). If severe enough, ΔΨ_(m) collapse and mitochondrial Ca²⁺sequestration can induce opening of a pore in the inner mitochondrialmembrane through a process called mitochondrial permeability transition(MPT), indirectly releasing cytochrome c and other proteins thatinitiate apoptosis (Bernardi et al., J Biol Chem 267:2934-2939, 1994;Zoratti and Szabo, Biochim Biophys Acta 1241:139-176, 1995; Ellerby etal., J Neurosci 17:6165-6178, 1997). Consistent with these observations,glutamate-induced excitotoxicity can be inhibited by preventingmitochondrial Ca²⁺ uptake or blocking MPT (Budd and Nichols, J Neurochem66:403-411, 1996; White and Reynolds, J Neurosci 16:5688-5697, 1996; Liet al., Brain Res 753:133-140,1997).

[0029] Whereas mitochondria-mediated apoptosis may be critical indegenerative diseases, it is thought that disorders such as cancerinvolve the unregulated and undesirable growth (hyperproliferation) ofcells that have somehow escaped a mechanism that normally triggersapoptosis in such undesirable cells. Enhanced expression of theanti-apoptotic protein, Bcl-2 and its homologues is involved in thepathogenesis of numerous human cancers. Bcl-2 acts by inhibitingprogrammed cell death and overexpression of Bcl-2, and the relatedprotein Bcl-xL, block mitochondrial release of cytochrome c frommitochondria and the activation of caspase 3 (Yang et al, Science275:1129-1132, 1997; Kluck et al., Science 275:1132-1136, 1997;Kharbanda et al., Proc. Natl. Acad. Sci. U.S.A. 94:6939-6942, 1997). Incontrast, overexpression of Bcl-2 and Bcl-xL protect against themitochondrial dysfunction preceding nuclear apoptosis that is induced bychemotherapeutic agents. In addition, acquired multi-drug resistance tocytotoxic drugs is associated with inhibition cytochrome c release thatis dependent on overexpression of Bcl-xL (Kojima et al., J. Biol. Chem.273: 16647-16650, 1998). Because mitochondria have been implicated inapoptosis, it is expected that agents that interact with mitochondrialcomponents will effect a cell's capacity to undergo apoptosis. Thus,agents that induce or promote apoptosis in hyperproliferative cells areexpected to be useful in treating hyperproliferative disorders anddiseases such as cancer.

[0030] Thus, alteration of mitochondrial function has great potentialfor a broad-based therapeutic strategy for designing drugs to treatdegenerative disorders and diseases as well as hyperproliferativediseases. Depending on the disease or disorder for which treatment issought, such drugs may be mitochondria protecting agents, anti-apoptoticagents or pro-apoptotic agents.

[0031] Clearly there is a need for compounds and methods that limit orprevent damage to organelles, cells and tissues by free radicalsgenerated intracellularly as a direct or indirect result ofmitochondrial dysfunction. In particular, because mitochondria areessential organelles for producing metabolic energy, agents that protectmitochondria against oxidative injury by free radicals would beespecially useful. Such agents may be suitable for the treatment ofdegenerative diseases including mitochondria associated diseases.Existing approaches to identifying agents that limit oxidative damagemay not include determination of whether such agents may help protectmitochondrial structure and/or function.

[0032] There is also a need for compounds and methods that limit orprevent damage to cells and tissues that occurs directly or indirectlyas a result of necrosis and/or inappropriate apoptosis. In particular,because mitochondria are mediators of apoptotic events. agents thatmodulate mitochondrially mediated pro-apoptotic events would beespecially useful. Such agents may be suitable for the treatment ofacute degenerative events such as stroke. Given the limited therapeuticwindow for blockade of necrotic death at the core of an infarct, it maybe particularly desirable to develop therapeutic strategies to limitneuronal death by preventing mitochondrial dysfunction in thenon-necrotic regions of an infarct. Agents and methods that maintainmitochondrial integrity during transient ischemia and the ensuing waveof excitotoxicity would be expected to be novel neuroprotective agentswith utility in limiting stroke-related neuronal injury.

[0033] There is also a need for compounds and methods that inhibit thegrowth or enhance the death of cells and tissues that have escapedappropriate apoptotic signals, as well as cytotoxic agents that causethe death of undesirable (e.g., cancer) cells by triggering theapoptotic cascade. In particular, because mitochondria are mediators ofapoptotic events, agents that stimulate mitochondrially mediatedpro-apoptotic events would be especially useful. Such agents may besuitable for the treatment of hyperproliferative diseases such as cancerand psoriasis.

[0034] The present invention fulfills these needs and provides otherrelated advantages. Those skilled in the art will recognize furtheradvantages and benefits of the invention after reading the disclosure.

SUMMARY OF THE INVENTION

[0035] Briefly stated, the present invention is directed to thetreatment of mitochondria-associated diseases by administration to awarm-blooded animal in need thereof an effective amount of a compoundhaving the following general structure (I):

[0036] where Ar is phenyl or naphthyl optionally substituted with 1 to 5R₂ groups and L is an optional linker moiety.

[0037] In one embodiment, Ar is phenyl, naphthyl, 4-bromonaphthyl,3,5-di-t-butyl-4-hydroxyphenyl, 2-methoxy-4-carboxylphenyl,2-chloro-4-carboxyl-5-methoxyphenyl, 3,5-di-tetrafluoromethylphenyl,3,5-difluorophenyl, 3,4,5-trimethoxyphenyl, 4-n-hexoxyphenyl,4-fluorophenyl, 3-trifluorophenyl, 2-carbinolphenyl,2-chloro-5-methylphenyl, 3-carboxylphenyl, 3-carboxyl-4-hydroxyphenyl,2-methyl-4-carboxylphenyl, 4-methoxyphenyl, 2-hydroxyphenyl,4-N-morphinol)phenyl, 3,4-dihydroxyphenyl, 2,4-dimethylphenyl,2-methyl-4-hydroxyphenyl, 4-n-octylphenyl, 2-hydroxy-5-n-octylphenyl,4-chlorophenyl, or 2-methyl-4-chlorophenyl.

[0038] In another embodiment the optional linker moiety L is notpresent, while in a further embodiment L is present and is —CH₂NH—,—CH₂CH₂, —CH(OH)CH₂—, —CH₂N(CH₃)— or —NHC(═NH)—.

[0039] In still further embodiments, methods are disclosed for treatingmitochondria-associated diseases by administering one or more compoundsof structure (I) in the form of a pharmaceutical composition. Thus,pharmaceutical compositions are also disclosed comprising a compound ofstructure (I) in combination with a pharmaceutically acceptable carrieror diluent.

[0040] In the context of this invention, mitochondria-associated diseaseinclude diseases in which free radical mediated oxidative injury leadsto tissue degeneration, diseases in which cells inappropriately undergoapoptosis, and diseases in which cells fail to undergo apoptosis. Thus,the methods of this invention include the treatment of a wide number ofmitochondria-associated diseases, including (but not limited toAlzheimer's Disease, Parkinson's Disease, Huntington's Disease,auto-immune disease, diabetes mellitus (Type I or Type II), congenitalmuscular dystrophy, fatal infantile myopathy, “later-onset” myopathy,MELAS (mitochondrial encephalopathy, lactic acidosis, and stroke), MIDD(mitochondrial diabetes and deafness), MERFF (myoclonic epilepsy raggedred fiber syndrome), arthritis, NARP (Neuropathy; Ataxia; RetinitisPigmentosa), MNGIE (Myopathy and external ophthalmoplegia; Neuropathy;Gastro-Intestinal; Encephalopathy), LHON (Leber's; Hereditary; Optic;Neuropathy), Kearns-Sayre disease, Pearson's Syndrome, PEO (ProgressiveExternal Ophthalmoplegia), Wolfram syndrome, DIDMOAD (DiabetesInsipidus, Diabetes Mellitus, Optic Atrophy, Deafness), Leigh'sSyndrome, dystonia. schizophrenia, cancer and psoriasis.

[0041] These and other aspects of the present invention will becomeevident upon reference to the following detailed description andattached drawings. In addition, various references are set forth hereinwhich describe in more detail certain aspects of this invention, and aretherefore incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWING

[0042] The attached FIGURE depicts attenuation of apoptosis in cellstreated with a representative compound of this invention, compound (11),prior to induction of an apoptotic pathway with ionophore.

DETAILED DESCRIPTION OF THE INVENTION

[0043] The present invention is directed generally to compounds usefulas mitochondria protecting agents, as well as methods useful fortreating mitochondria associated diseases. More specifically, themitochondria protecting agents of this invention have the followingstructure (I):

[0044] including stereoisomers, prodrugs and pharmaceutically acceptablesalts thereof,

[0045] wherein:

[0046] Ar is phenyl or naphthyl optionally substituted with 1 to 5 R₂groups;

[0047] L is an optional linker moiety selected from —(CH₂)_(n)—,—(CH₂)_(n)NH—, —(CH₂)_(n)N(C₁₋₄alkyl)—, —NHC(═NH)— and—(CH₂)_(n)O(CH₂)_(n)—, wherein n is 1-4 and each linker moiety isoptionally substituted with 1 to 5 R₃ groups;

[0048] R₂ is hydroxy, C₁₋₂alkyl, C₁₋₁₂alkyloxy, halo, —NH₂, —NHR, —NRR,cyano, nitro, —SR, —COOH, C₇₋₁₂aralkyl or heterocycle; or C₁₋₁₂alkyl,C₁₋₁₂alkyloxy, —NH₂, —NHR, —NRR, —SR, C₇₋₁₂aralkyl or heterocyclesubstituted with 1 to 5 R₃ groups;

[0049] R₃ is hydroxy, halo, C₁₋₄alkyl, —OR, —NH₂, —NHR or —NRR; and

[0050] each occurrence of R is independently selected from C₁₋₄alkyl.

[0051] As used herein, a “C₁₋₄ alkyl” is a straight chain or branched,saturated or unsaturated hydrocarbon moiety having from 1 to 4 carbonatoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, andthe like. Similarly, “C₁₋₁₂alkyl” is a straight chain or branched,saturated or unsaturated hydrocarbon moiety having from 1 to 12 carbonatoms, including the above C₁₋₄ alkyls as well as n-pentyl, n-octyl andthe like, and branched hydrocarbons such as1,1-dimethyl-3,3-dimethyl-butyl and the like.

[0052] “C₁₋₁₂alkyloxy” means —O— C₁₋₁₂alkyl, such as methoxy, ethoxy andthe like.

[0053] “Halo” means fluoro, chloro, bromo or iodo.

[0054] “C₇₋₁₂aralkyl” refers to a moiety having both an aryl and alkylportion, wherein the combined numbers of carbon atoms for both portionsrange from 7 to 12. As used herein, “aryl” refers to aromatic monocyclicand fused, homoaryl and heteroaryl groups. “Homoaryl” refers to anaromatic compound having an aromatic ring made up of only carbon atoms,while the term “heteroaryl” refers to an aromatic compound having anaromatic ring which contains, in addition to carbon, one or more otheratoms, most commonly nitrogen, oxygen and sulfur. The term “monocyclicaryl” refers to an aromatic compound having a single aromatic ring,while “fused aryl” refers to aromatic rings that shares a pair of carbonatoms, and includes multiple fused rings. Representative C₇₋₁₂aralkylmoieties include, but are not limited to, benzyl and —C(CH₃)₂-phenyl.

[0055] “Heterocycle” means a 5- to 7-membered monocyclic or 7- to10-membered bicyclic heterocycle ring which is either saturated orunsaturated, and which contains carbon atoms and from 1 to 4 heteroatomsselected from N, O and S, wherein the N and S heteroatoms may beoptionally oxidized, and wherein the N heteroatom may be optionallyquaternized. The heterocycle may be attached via any carbon atom orheteroatom on the ring. Representative heterocycles include, forexample, morpholine.

[0056] The phrase “substituted C₁₋₁₂alkyl, C₁₋₁₂alkyloxy, —NH₂, —NHR,—NRR, —SR, C₇₋₁₂aralkyl or heterocycle substituted with 1 to 5 R₃groups” means that from one to five hydrogen atoms of the C₁₋₁₂alkyl,C₁₋₁₂alkyloxy, —NH₂, —NHR, —NRR, —SR, C₇₋₁₂aralkyl or heterocycle moietyhave been replaced with a R₃ group, wherein each R₃ group may be thesame or different. For example, representative substituted C₁₋₁₂alkylsinclude trifluromethyl and —CH₂OH.

[0057] Similarly, a “substituted” linker moiety is when from one to fivehydrogen atoms of —(CH₂)_(n)—, —(CH₂)_(n)NH—, —(CH₂)_(n)N(C₁₋₄alkyl)—,—NHC(═NH)— or —(CH₂)_(n)O(CH₂)_(n)— have been replaced with a R₃ group,wherein each R₃ group may be same or different. For example,representative substituted linkers include —CH(OH)CH₂— when the linkeris —CH₂CH₂— substituted with a hydroxyl R₃ group.

[0058] Representative compounds of this invention and analytical datafor the same are presented in the following Tables 1 and 2. TABLE 1Representative Compounds

Cpd. Ar L  (1)

—CH₂NH—  (2)

—(CH₂)₂—  (3)

(none)  (4)

 (5)

—(CH₂)₂—  (6)

(none)  (7)

(none)  (8)

(none)  (9)

(none) (10)

(11)

(12)

(none) (13)

(none) (14)

(none) (15)

(none) (16)

(none) (17)

(none) (18)

(none) (19)

(none) (20)

(none) (21)

(none) (22)

(none) (23)

(none) (24)

(none) (25)

—CH₂NH— (26)

—CH₂NH— (27)

(none) (28)

—CH₂CH₂— (29)

(none) (30)

(none) (31)

(none) (32)

(none) (33)

(none) (34)

(35)

[0059] TABLE 2 Analytical Data Cpd. ¹H NMR (500 MHz) MW^(†)  (1)(acetate salt in CD₃OD): δ 7.14 (s, 352 2H), 3.82 (s, 2H), 1.97 (acetateCH₃ 293.2 (GH)⁺ peak), 1.42 (s, 18H)  (2) (acetate salt in CD₃OD): δ7.07 (d, 2H, J = 235 8.4 Hz), 6.74 (d, 2H, J = 8.4 Hz), 3.39 223.2 (GH)⁺(t, 2H, J = 7 Hz), 2.78 (t, 2H, J = 7 Hz), 1.97 (acetate CH₃ peak).  (3)(in CD₃OD): δ 6.59 (s, 2H), 3.84 (s, 6H), 225 3.77 (s, 3H) 226 (GH)⁺ (4) (in CD₃OD): δ 7.11 (s, 2H), 3.87 (d, 1H), 306 3.58 (d, 1H), 2.68(s, 3H), 1.41 (s, 18H) 308.2 (GH₂)⁺  (5) (acetate salt in CD₃OD): δ 6.89(d, 1H, J = 283 8.1 Hz), 6.87 (d, 1H, 1.9 Hz), (dd, 1H, 179.2 (GH)⁺ J =8.1, 1.9 Hz), 3.83 (s, 3H), 3.80 (s, 3H), 3.43 (t, 2H, J = 7.1 Hz), 2.82(t, 2H, J = 7.1 Hz), 1.97 (acetate CH₃ peak).  (6) (in CD₃OD): δ 7.04(dd, 1H, J = 2.1, 8.3 263 Hz), 6.98 (d, 1H, J = 2.1 Hz), 6.87 (d, 264.1(GH)⁺ 1H, J = 8.3 Hz), 2.54 (t, 2H, 7.6 Hz), 1.58 (br. t, 2H), 1.30 (m,10 H), 0.89 (t, 3H, J = 7 Hz).  (7) (acetate salt in CD₃OD): δ 7.93 (dd,1H, 323 J = 6.6, 2.1 Hz), 8.01 (m, 1H), 7.92 (d, 263.9 (G)⁺ 1H, J = 8Hz), 7.74 (m, 2H), 7.43 (d, 7.7 Hz), 1.98 (acetate CH₃ peak)  (9)(acetate salt in CD₃OD): δ 7.47 (d, 2H, J = 229 8.6 Hz), 7.27 (d, 2H, J= 8.6 Hz), 1.95 170.1 (GH)⁺ (acetate CH₃ peak) (10) (in CD₃OD): δ 7.42(d, 2H), 7.37 (m, 179 2H), 7.30 (m, 1H), 4.83 (m, 1H), 3.43 179.9 (G)⁺(dd, 1H, J = 13.7, 3.7 Hz) 3.35 (dd, 1H, 13.9, 7.5Hz) (11) (HCl salt inCD₃OD): δ 7.33 (d, 4H), 213.5 7.14 (m, 1H) 177.8 (GH)⁺ (12) (acetatesalt in CD₃OD): δ 8.15 (d, 1H, J = 269 8.2 Hz), 7.49 (d, 1H, J = 1.6Hz), 7.46 208.0 (G-H)⁺ (dd, 1H, J = 8.2, 1.6 Hz), 3.95 (s, 3H), 1.93(acetate CH₃ peak) (13) (in CD₃OD): δ 8.18 (s, 1H), 7.97 (s, 1H), 2433.97 (s, 3H) 244.2 (GH)⁺ (14) (acetate salt in CD₃OD): δ 7.92 (s, 1H),331 7.89 (s, 2H), 1.97 (acetate CH₃ peak) 271.9 (GH)⁺ (15) (in CD₃OD): δ6.94 (d, 1H), 6.93 (d, 2H) 171 172 (GH)⁺ (16) (acetate salt in CD₃OD): δ7.18 (d, 2H, J = 295 8.8 Hz), 6.99 (d, 2H, J = 8.8 Hz), 3.99 236.0 (GH)⁺(t, 2H, J = 6.5 Hz), 1.98 (acetate CH₃ peak), 1.77 (m, 2H), 1.47 (m,2H), 1.36 (m, 4H), 0.92 (t, 3H, 6.8 Hz) (17) (acetate salt in CD₃OD):δ7.31 (m, 2H), 213 7.20 (m, 2H) 1.97 (acetate CH₃ peak) 154.0 (GH)⁺ (18)(acetate salt in CD₃OD): δ 7.55-7.68 (m, 263 4H), 1.96 (acetate CH₃peak) 204.0 (GH)⁺ (19) (acetate salt in CD₃OD): δ 7.55 (m, 1H), 225 7.41(m, 2H), 7.30 (m, 2H), 4.63 (s, 2H), 166.0 (GH)⁺ 1.98 (acetate CH₃ peak)(20) (in CD₃OD): δ 7.47 (m, 1H), 7.36 (m, 135 2H), 7.29 (m, 2H) 136.1(GH)⁺ (21) (acetate salt in CD₃OD): δ 7.45 (d, 1H, J = 243 8.1 Hz), 7.24(m, 2H), 2.36 (s, 3H), 184 (GH)⁺ 1.95 (acetate CH₃ peak) (22) (inCD₃OD): δ 7.84 (m, 1H), 7.55 (dd, 179 1H, J = 8.1, 2.1 Hz), 7.46 (m,1H), 7.34 202.2 (m, 1H) (G + Na)⁺ (23) (acetate salt in CD₃OD): δ 7.76(d, 1H, J = 255 2.6 Hz), 7.37 (dd, 1H, 8.8, 2.6 Hz), 196 (GH)⁺ 7.02 (d,1H, 8.8 Hz), 1.99 (acetate CH₃ peak) (24) (in CD₃OD): δ 7.84 (d, 1H, J =8.5 Hz), 193 7.07 (br. S, 1H), 7.66 (dd, 1H, J = 8.5, 216.2 2.1 Hz),2.30 (3H) (G + Na)⁺ (25) (acetate salt in CD₃OD): δ 7.28 (d, 2H, J = 2548.6 Hz), 6.89 (d, 2H, J = 8.6 Hz), 3.85 195.3 (GH)⁺ (s, 2H), 3.77 (s,3H), 1.93 (acetate CH₃ peak) (26) (acetate salt in CD₃OD): δ 7.16 (m,2H), 240 6.79 (m, 2H), 3.93 (s, 2H), 1.93 (acetate 181.2 (GH)⁺ CH₃ peak)(27) (acetate salt in CD₃OD): δ 7.16 (d, 2H, J = 280 9 Hz), 7.05 (d, 2H,J = 9 Hz), 3.83 (m, 221.2 (GH)⁺ 4H), 3.18 (m, 2H), 1.98 (acetate CH₃peak) (28) (acetate salt in CD₃OD): δ 6.7 (d, 1H, J = 255 8 Hz), 6.66(d, 1H, 1.9 Hz), 6.55 (dd, 1H, 196.1 (GH)⁺ J = 8, 1.9 Hz), 3.37 (t, 2H,J = 7 Hz),, 2.72 (t, 2H, J = 7 Hz), 1.99 (acetate CH₃ peak) (29)(acetate salt in CD₃OD): δ 7.18 (br. S, 223 1H), 7.10 (br. T, 2H), 2.33(s, 3H), 2.25 164.2 (GH)⁺ (s., 3H), 1.98 (acetate CH₃ peak) (30)(acetate salt in CD₃OD): δ 7.03 (d, 1H, J = 225 8.5 Hz), 6.76 (d, 1H, J= 2.7 Hz), 6.69 166.3 (GH)⁺ (dd, 1H, J = 8.5, 2,7 Hz), 2.20 (s, 3H),1.98 (acetate CH₃ peak) (31) (in CD₃OD): δ 7.29 (br. d, 2H, J = 8.2 247Hz), 7.17 (dd, 2H, J = 6.7, 1.8 Hz), 2.64 248.4 (GH)⁺ (t, 2H, J = 7.6Hz), 1.62 (m, 2H), 1.30 (m, 10H), 0.89 (t, 3H, J = 6.7 Hz) (32) (acetatesalt in CD₃OD): δ 7.48 (dd, 1H, 243 J = 8.0, 1.0 Hz), 7.29 (t, 1H), 7.21(br. d, 183.9 (GH)⁺ 1H), 2.40 (s, 3H), 1.93 (acetate CH₃ peak) (33)(acetate salt in CD₃OD): δ 7.39 (d, 1H, 243 2.2 Hz), 7.30 (dd, 1H, J =8.6, 2.2 Hz), 183.9 (GH)⁺ 7.22 (d, 1H, 8.3 Hz), 2.28 (s, 3H), 1.98(acetate CH₃ peak) (34) (in CD₃OD): δ 7.97 (d, 2H), 7.49 (d, 2H) 222 222(GH)⁺ (35) (in CD₃OD): δ 7.97 (d, 2H), 7.49 (d, 2H) 222 222 (GH)⁺

[0060] The compounds of the present invention may be prepared by knownorganic synthesis techniques, including the methods described in moredetail in the Examples. In general, the compounds of this invention maybe prepared by the following reaction scheme:

[0061] Pharmaceutically acceptable salts of the compounds of thisinvention may be made by techniques well known in the art, such as byreacting the free acid or base forms of these compounds with astoichiometric amount of the appropriate base or acid in water of in anorganic solvent. Suitable salts in this context may be found inRemington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co.,Easton, Pa., 1985, which is hereby incorporated by reference.

[0062] By way of example and not limitation, suitable pharmaceuticallyacceptable salts of the compounds of this invention include acidaddition salts which may, for example, be formed by mixing a solution ofthe compound according to the invention with a solution of an acceptableacid such as hydrobromic acid, hydrochloric acid, fumaric acid, oxalicacid, p-toluenesulphonic acid, malic acid, maleic acid, methanesulfonicacid, succinic acid, acetic acid, citric acid, tartaric acid, carbonicacid, phosphoric acid, sulphuric acid and the like. The salts may beformed by conventional means, such as by reacting the free base form ofthe product with one or more equivalents of the appropriate acid in asolvent or medium in which the salt is insoluble, or in a solvent suchas water which is removed in vacuo or by freeze drying or by exchangingthe anions of an existing salt for another anion on a suitable ionexchange resin. By way of example and not limitation, suitablepharmaceutically acceptable salts of the compounds of this inventioninclude acid addition salts which may, for example, be formed by mixinga solution of the compound according to the invention with a solution ofan acceptable acid such as hydrobromic acid, hydrochloric acid, fumaricacid, oxalic acid, p-toluenesulphonic acid, malic acid, maleic acid,methanesulfonic acid, succinic acid, acetic acid, citric acid, tartaricacid, carbonic acid, phosphoric acid, sulphuric acid and the like. Thesalts may be formed by conventional means, such as by reacting the freebase form of the product with one or more equivalents of the appropriateacid in a solvent or medium in which the salt is insoluble, or in asolvent such as water which is removed in vacuo or by freeze drying orby exchanging the anions of an existing salt for another anion on asuitable ion exchange resin.

[0063] A compounds of this invention, or a pharmaceutically acceptablesalt thereof, is administered to a patient in a therapeuticallyeffective amount. A therapeutically effective amount is an amountcalculated to achieve the desired effect. It will be apparent to oneskilled in the art that the route of administration may vary with theparticular treatment. Routes of administration may be eithernon-invasive or invasive. Non-invasive routes of administration includeoral, buccal/sublingual, rectal, nasal, topical (including transdermaland ophthalmic), vaginal, intravesical, and pulmonary. Invasive routesof administration include intraarterial, intravenous, intradermal,intramuscular, subcutaneous, intraperitoneal, intrathecal andintraocular.

[0064] The required dosage may vary with the particular treatment androute of administration. In general, dosages for mitochondria protectingagents will be from about 1 to about 5 milligrams of the compound perkilogram of the body weight of the host animal per day; frequently itwill be between about 100 μg and about 5 mg but may vary up to about 50mg of compound per kg of body weight per day. Therapeutic administrationis generally performed under the guidance of a physician, andpharmaceutical compositions contain the mitochondria protecting agent ina pharmaceutically acceptable carrier. These carriers are well known inthe art and typically contain non-toxic salts and buffers. Such carriersmay comprise buffers like physiologically-buffered saline,phosphate-buffered saline, carbohydrates such as glucose, mannose,sucrose, mannitol or dextrans, amino acids such as glycine,antioxidants, chelating agents such as EDTA or glutathione, adjuvantsand preservatives. Acceptable nontoxic salts include acid addition saltsor metal complexes, e.g., with zinc, iron, calcium, barium. magnesium,aluminum or the like (which are considered as addition salts forpurposes of this application). Illustrative of such acid addition saltsare hydrochloride, hydrobromide, sulphate, phosphate, tannate, oxalate,fumarate, gluconate, alginate, maleate, acetate, citrate, benzoate,succinate, malate, ascorbate, tartrate and the like. If the activeingredient is to be administered in tablet form, the tablet may containa binder, such as tragacanth, corn starch or gelatin; a disintegratingagent, such as alginic acid; and a lubricant, such as magnesiumstearate. If administration in liquid form is desired, sweetening and/orflavoring may be used, and intravenous administration in isotonicsaline, phosphate buffer solutions or the like may be effected.

[0065] In one embodiment of the invention, pharmaceutical compositionscomprising one or more compounds of this invention are entrapped withinliposomes. Liposomes are microscopic spheres having an aqueous coresurrounded by one or more outer layer(s) made up of lipids arranged in abilayer configuration (see, e.g., Chonn et al., Current Op. Biotech.6:698, 1995). The therapeutic potential of liposomes as drug deliveryagents was recognized nearly thirty years ago (Sessa et al., J. LipidRes. 9:310, 1968). Liposomes include “sterically stabilized liposome,” aterm which, as used herein, refers to a liposome comprising one or morespecialized lipids that, when incorporated into liposomes, result inenhanced circulation lifetimes relative to liposomes lacking suchspecialized lipids. Examples of sterically stabilized liposomes arethose in which part of the vesicle-forming lipid portion of the liposome(A) comprises one or more glycolipids, such as monosialogangliosideG_(M1), or (B) is derivatized with one or more hydrophilic polymers,such as a polyethylene glycol (PEG) moiety. While not wishing to bebound by any particular theory, it is thought in the art that, at leastfor sterically stabilized liposomes containing gangliosides,sphingomyelin, or PEG-derivatized lipids, the enhanced circulationhalf-life of these sterically stabilized liposomes derives from areduced uptake into cells of the reticuloendothelial system (RES) (Allenet al., FEBS Letters 223:42, 1987; Wu et al., Cancer Research 53:3765,1993).

[0066] Various liposomes comprising one or more glycolipids are known inthe art. Papahadjopoulos et al. (Ann. N.Y Acad. Sci., 507:64, 1987)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A. 85:6949, 1988). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al.).

[0067] Various liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn. 53:2778, 1980) describedliposomes comprising a nonionic detergent, 2C, 215G, that contains a PEGmoiety. Illum et al. (FEBS Letters 167:79, 1984) noted that hydrophiliccoating of polystyrene particles with polymeric glycols results insignificantly enhanced blood half-lives. Synthetic phospholipidsmodified by the attachment of carboxylic groups of polyalkylene glycols(e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and4,534,899). Klibanov et al. (FEBS Letts. 268:235, 1990) describedexperiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta 1029:91, 1990) extended such observationsto other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from thecombination of distearoylphosphatidylethanolamine (DSPE) and PEG.Liposomes having covalently bound PEG moieties on their external surfaceare described in European Patent No. 0 445 131 B1 and WO 90/04384 toFisher. Liposome compositions containing 1-20 mole percent of PEderivatized with PEG, and methods of use thereof, are described byWoodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al.(U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1).Liposomes comprising a number of other lipid-polymer conjugates aredisclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin etal.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).U.S. Pat. Nos. 5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.)describe PEG-containing liposomes that can be further derivatized withfunctional moieties on their surfaces.

[0068] When the compounds of the invention are prepared to treat chronicneurological disorders (such as, e.g., Alzheimer's disease) or acutenecrotic events (such as, e.g., stroke), one preferred pharmaceuticalcomposition is one in which a compound of the invention is encapsulatedwithin a PEG-containing liposome that has been derivatized to include afactor that targets the liposome and its contents a portion of thecentral nervous system (CNS), such as, for example, the brain. Such afactor may be attached to the lipid bilayer of the liposome or to a PEGmoiety that is incorporated into the liposome. By way of example and notlimitation, one brain-targeting factor that can be used withPEG-containing liposomes is an antibody to a receptor that mediatesuptake of one or more peptides through the blood brain barrier (BBB).Such peptides include, for example, insulin, insulin-like growthfactors, transferrin and leptin. The antibody of the PEG-containingliposome, which may be a monoclonal antibody, targets the liposome andits contents to the brain via a specific interaction with a BBB peptidereceptor such as, e.g., the BBB transferrin receptor (Huwyler et al.,Proc. Natl. Acad. Sci. U.S.A. 93:14164-14169, 1996).

[0069] Mitochondria protecting agents of this invention also includeprodrugs thereof. As used herein, a “prodrug” is any covalently bondedcarrier that releases in vivo the active parent drug according thestructure (I) when such prodrug is administered to the animal. Prodrugsof the compounds of structure (I) are prepared by modifying functionalgroups present on the compound in such a way that the modifications arecleaved, either in routine manipulation or in vivo, to the parentcompound. Prodrugs include, but are not limited to, compounds ofstructure (I) wherein hydroxy, amine or sulfhydryl groups are bonded toany group that, when administered to the animal, cleaves to form thefree hydroxyl, amino or sulfhydryl group, respectively. Representativeexamples of prodrugs include, but are not limited to, acetate, formateand benzoate derivatives of alcohol and amine functional groups.

[0070] The effectiveness of a compound as a mitochondria protectingagent may be determined by various assay methods. Suitable mitochondriaprotecting agents of this invention are active in one or more of thefollowing assays for maintenance of mitochondrial structural andfunctional integrity, or in any other assay known in the art thatmeasures the maintenance of mitochondrial structural and functionalintegrity. Accordingly, it is an aspect of the invention to providemethods for treating mitochondria associated diseases that includemethods of administering compounds that may or may not have knownantioxidant properties. However, according to this aspect of theinvention, the unexpected finding is disclosed herein that mitochondriaprotecting agents may exhibit mitochondria protecting activities thatare not predictable based upon determination of antioxidant propertiesin non-mitochondrial assay systems.

[0071] A. Assay for Inhibition of Production of Reactive Oxygen SpeciesUsing Dichlorofluorescin Diacetate

[0072] According to this assay, the ability of a mitochondria protectingagent of the invention to inhibit production of ROS intracellularly maybe compared to its antioxidant activity in a cell-free environment.Production of ROS may be monitored using, for example by way ofillustration and not limitation, 2′,7′-dichlorodihydrofluresceindiacetate (“dichlorofluorescin diacetate” or DCFC), a sensitiveindicator of the presence of oxidizing species. Non-fluorescent DCFC isconverted upon oxidation to a fluorophore that can be quantifiedfluorimetrically. Cell membranes are also permeable to DCFC, but thecharged acetate groups of DCFC are removed by intracellular esteraseactivity, rendering the indicator less able to diffuse back out of thecell.

[0073] In the cell-based aspect of the DCFC assay for inhibition ofproduction of ROS, cultured cells may be pre-loaded with a suitableamount of DCFC and then contacted with a mitochondria protecting agent.After an appropriate interval, free radical production in the culturedcells may be induced by contacting them with iron (III)/ascorbate andthe relative mean DCFC fluorescence can be monitored as a function oftime.

[0074] In the cell-free aspect of the DCFC assay for inhibition ofproduction of ROS, a mitochondria protecting agent may be tested for itsability to directly inhibit iron/ ascorbate induced oxidation of DCFCwhen the protecting agent, the fluorescent indicator and the freeradical former are all present in solution in the absence of cells.

[0075] Comparison of the properties of a mitochondria protecting agentin the cell-based and the cell-free aspects of the DCFC assay may permitdetermination of whether inhibition of ROS production by a mitochondriaprotecting agent proceeds stoichiometrically or catalytically. Withoutwishing to be bound by theory, mitochondria protecting agents thatscavenge free radicals stoichiometrically (e.g., on a one-to-onemolecular basis) may not represent preferred agents because highintracellular concentrations of such agents might be required for themto be effective in vivo. On the other hand, mitochondria protectingagents that act catalytically may moderate production of oxygen radicalsat their source, or may block ROS production without the agentsthemselves being altered, or may alter the reactivity of ROS by anunknown mechanism. Such mitochondria protecting agents may “recycle” sothat they can inhibit ROS at substoichiometric concentrations.Determination of this type of catalytic inhibition of ROS production bya mitochondria protecting agent in cells may indicate interaction of theagent with one or more cellular components that synergize with the agentto reduce or prevent ROS generation. A mitochondria protecting agenthaving such catalytic inhibitory characteristics may be a preferredagent for use according to the method of the invention.

[0076] Mitochondria protecting agents that are useful according to theinstant invention may inhibit ROS production as quantified by thisfluorescence assay or by other assays based on similar principles. Theperson having ordinary skill in the art is familiar with variations andmodifications that may be made to the assay as described here withoutdeparting from the essence of this method for determining theeffectiveness of a mitochondria protecting agent, and such variationsand modifications are within the scope of this disclosure.

[0077] B. Assay for Mitochondrial Permeability Transition (MPT) Using2-,4-Dimethvlaminostyryl-N-Methvlpyridinium (DASPMI).

[0078] According to this assay, one may determine the ability of amitochondria protecting agent of the invention to inhibit the loss ofmitochondrial membrane potential that accompanies mitochondrialdysfunction. As noted above, maintenance of a mitochondrial membranepotential may be compromised as a consequence of mitochondrialdysfunction. This loss of membrane potential or mitochondrialpermeability transition (MPT) can be quantitatively measured using themitochondria-selective fluorescent probe2-,4-dimethylaminostyryl-N-methylpyridinium (DASPMI).

[0079] Upon introduction into cell cultures, DASPMI accumulates inmitochondria in a manner that is dependent on, and proportional to,mitochondrial membrane potential. If mitochondrial function is disruptedin such a manner as to compromise membrane potential, the fluorescentindicator compound leaks out of the membrane bounded organelle with aconcomitant loss of detectable fluorescence. Fluorimetric measurement ofthe rate of decay of mitochondria associated DASPMI fluorescenceprovides a quantitative measure of loss of membrane potential, or MPT.Because mitochondrial dysfunction may be the result of reactive freeradicals such as ROS, mitochondria protecting agents that retard therate of loss of DASPMI fluorescence may be effective agents for treatingmitochondria associated diseases according to the methods of the instantinvention.

[0080] C. Assays of Apoptosis in Cells Treated with MitochondriaProtecting Agents

[0081] As noted above, mitochondrial dysfunction may be an inductionsignal for cellular apoptosis. According to the assays in this section,one may determine the ability of a mitochondria protecting agent of theinvention to inhibit or delay the onset of apoptosis. Mitochondrialdysfunction may be present in cells known or suspected of being derivedfrom a subject with a mitochondria associated disease, or mitochondrialdysfunction may be induced in cultured normal or diseases cells by oneor more of a variety of physical (e.g., UV radiation), physiological andbiochemical stimuli with which those having skill in the art will befamiliar.

[0082] Apoptosis and/or biochemical processes associated with apoptosismay also be using one or more “apoptogens,” i.e., agents that induceapoptosis and/or associated processes when contacted with or withdrawnfrom cells or isolated mitochondria. Such apoptogens include by way ofillustration and not limitation (1) apoptogens that are added to cellshaving specific receptors therefor, e.g., tumor necrosis factor (TNF),FasL, glutamate and NMDA; (2) withdrawal of growth factors from cellshaving specific receptors for such factors, such factors including, forexample, IL-3 or corticosterone; and apoptogens that may be added tocells but which do not require a specific receptor, including (3)Herbimycin A (Mancini et al., J. Cell. Biol. 138:449-469, 1997), (4)Paraquat (Costantini et al., Toxicology 99:1-2, 1995); (5) ethyleneglycols (http://www.ulaval.ca/vrr/rech/Proj/532866.html); (6) proteinkinase inhibitors, such as, e.g.: Staurosporine, Calphostin C,d-erythro-sphingosine derivatives, Chelerythrine chloride. Genistein,1-(5-isoquinolinesulfonyl)-2-methylpiperazine, KN-93, Quercitin,N-[2-((p-bromocinnamyl)amino)ethyl]-5-5-isoquinolinesulfonamide andcaffeic acid phenethyl ester; (7) ionophores such as, e.g.: lonomycinand valinomycin; (8) MAP kinase inducers such as, e.g.: Anisomycin andAnandamine; (9) cell cycle blockers such as, e.g.: Aphidicolin,Colcemid, 5-fluorouracil and homoharringtonine; (10)Acetylcholineesterase inhibitors such as, e.g.: berberine; (11)anti-estrogens such as, e.g.: Tamoxifen; (12) pro-oxidants, such as,e.g., tert-butyl peroxide and hydrogen peroxide; (13) free radicals suchas, e.g., nitrous oxide; (14) inorganic metal ions, such as, e.g.:cadmium; (15) DNA synthesis inhibitors such as, for example, ActinomycinD, Bleomycin sulfate, Hydroxyurea, Methotrexate, Mitomycin C,Camptothecin, daunorubicin and intercalators such as, e.g., doxorubicin;(16) protein synthesis inhibitors such as, e.g., cyclohexamide,puromycin and rapamycin; (17) agents that affect microtubulin formationor stability such as, e.g., Vinblastine, Vincristine, colchicine,4-hydroxyphenylretinarnide and paclitaxel; (18) agents that raiseintracellular calcium levels by causing the release thereof fromintracellular stores, such as, e.g., thapsigargin (Thastrup et al.,Proc. Natl. Acad. Sci. U.S.A. 87:2466-2470, 1990) and thpasigargicin(Santarius et al., Toxicon 25:389-399, 1987); and agents that are addedto isolated mitochondria such as (19) MPT inducers, e.g., Bax protein(Jurgenmeier et al., Proc. Natl. Acad. Sci. U.S.A. 95:4997-5002, 1998);and (20) calcium and inorganic phosphate (Kroemer et al., Ann. Rev.Physiol 60:619, 1998).

[0083] In one aspect of the apoptosis assays, cells that are suspectedof undergoing apoptosis may be examined for morphological, permeabilityor other changes that are indicative of an apoptotic state. For exampleby way of illustration and not limitation, apoptosis in many cell typesmay cause altered morphological appearance such as plasma membraneblebbing, cell shape change, loss of substrate adhesion properties orother morphological changes that can be readily detected by thoseskilled in the art using light microscopy. As another example, cellsundergoing apoptosis may exhibit fragmentation and disintegration ofchromosomes, which may be apparent by microscopy and/or through the useof DNA specific or chromatin specific dyes that are known in the art,including fluorescent dyes. Such cells may also exhibit altered membranepermeability properties as may be readily detected through the use ofvital dyes (e.g., propidium iodide, trypan blue) or the detection oflactate dehydrogenase leakage into the extracellular milieu. Damage toDNA may also be assayed using electrophoretic techniques (see, forexample, Morris et al., BioTechniques 26:282-289, 1999). These and othermeans for detecting apoptotic cells by morphologic, permeability andrelated changes will be apparent to those familiar with the art.

[0084] In another aspect of the apoptosis assays, translocation of cellmembrane phosphatidylserine (PS) from the inner to the outer leaflet ofthe plasma membrane is quantified by measuring outer leaflet binding bythe PS-specific protein annexin (Martin et al, J. Exp. Med. 182:1545,1995; Fadok et al., J. Immunol. 148:2207. 1992.). In a perferred format,exteriorization of plasma membrane PS is assessed in 96 well platesusing a labeled annexin derivative such as an annexin-fluoresceinisothiocyanate conjugate (annexin-FITC, Oncogene Research Products,Cambridge, Mass.).

[0085] In another aspect of the apoptosis assays, quantification of themitochondrial protein cytochrome c that has leaked out of mitochondriain apoptotic cells may provide an apoptosis indicator that can bereadily determined (Liu et al., Cell 86:147-157, 1996). Suchquantification of cytochrome c may be performed spectrophotometrically,immunochemically or by other well established methods for detecting thepresence of a specific protein. Release of cytochrome c frommitochondria in cells challenged with apoptotic stimuli (e.g.,ionomycin, a well known calcium ionophore) can be followed by a varietyof immunological methods. Matrix-assisted laser desorption ionizationtime of flight mass (MALDI-TOF) spectrometry coupled with affinitycapture is particularly suitable for such analysis since apo-cytochromec and holo cytochrome c can be distinguished on the basis of theirunique molecular weights. For example, the SELDI system (Ciphergen, PaloAlto, USA) may be utilized to follow the inhibition by mitochondriaprotecting agents of cytochrome c release from mitochondria in ionomycintreated cells. In this approach, a cytochrome c specific antibodyimmobilized on a solid support is used to capture released cytochrome cpresent in a soluble cell extract. The captured protein is then encasedin a matrix of an energy absorption molecule (EAM) and is desorbed fromthe solid support surface using pulsed laser excitation. The molecularweight of the protein is determined by its time of flight to thedetector of the SELDI mass spectrometer.

[0086] In another aspect of the apoptosis assays, induction of specificprotease activity in a family of apoptosis-activated proteases known asthe caspases (Thomberry and Lazebnik, Science 281:1312-1316, 1998) ismeasured, for example by determination of caspase-mediated cleavage ofspecifically recognized protein substrates. These substrates mayinclude, for example, poly-(ADP-ribose) polymerase (PARP) or othernaturally occurring or synthetic peptides and proteins cleaved bycaspases that are known in the art (see, e.g., Ellerby et al., 1997 J.Neurosci. 17:6165). The labeled synthetic peptide Z-Tyr-Val-Ala-Asp-AFC,wherein “Z” indicates a benzoyl carbonyl moiety and AFC indicates7-amino-4-trifluoromethylcoumarin (Kluck et al., 1997 Science 275:1132;Nicholson et al., 1995 Nature 376:37), is one such substrate. Anotherlabeled synthetic peptide substrate for caspase-3 consists of twofluorescent proteins linked to each other via a peptide linkercomprising the recognition/cleavage site for the protease (Xu et al.,Nucleic Acids Res. 26:2034-2035, 1998). Other substrates include nuclearproteins such as U 1-70 kDa and DNA-PKcs (Rosen and Casciola-Rosen, 1997J. Cell. Biochem. 64:50; Cohen, 1997 Biochem. J. 326:1).

[0087] In another aspect of the apoptosis assays, the ratio of living todead cells, or the proportion of dead cells, in a population of cellsexposed to an apoptogen is determined as a measure of the ultimateconsequence of apoptosis. Living cells can be distinguished from deadcells using any of a number of techniques known to those skilled in theart. By way of non-limiting example, vital dyes such as propidium iodideor trypan blue may be used to determine the proportion of dead cells ina population of cells that have been treated with an apoptogen and acompound according to the invention (see Example 7).

[0088] The person of ordinary skill in the art will readily appreciatethat there may be other suitable techniques for quantifying apoptosis,and such techniques for purposes of determining the effects ofmitochondria protecting agents on the induction and kinetics ofapoptosis are within the scope of the assays disclosed here.

[0089] D. Assay of Electron Transport Chain (ETC) Activity in IsolatedMitochondria.

[0090] As described above, mitochondria associated diseases may becharacterized by impaired mitochondrial respiratory activity that may bethe direct or indirect consequence of elevated levels of reactive freeradicals such as ROS. Accordingly, a mitochondria protecting agent foruse in the methods provided by the instant invention may restore orprevent further deterioration of ETC activity in mitochondria ofindividuals having mitochondria associated diseases. Assay methods formonitoring the enzymatic activities of mitochondrial ETC Complexes I,II, III, IV and ATP synthetase, and for monitoring oxygen consumption bymitochondria, are well known in the art. (See, e.g, Parker et al.,Neurology 44:1090-96, 1994; Miller et al, J. Neurochem. 67:1897, 1996.)It is within the scope of the methods provided by the instant inventionto identify a mitochondria protecting agent using such assays ofmitochondrial function. Further, mitochondrial function may be monitoredby measuring the oxidation state of mitochondrial cytochrome c at 540nm. As described above, oxidative damage that may arise in mitochondriaassociated diseases may include damage to mitochondrial components suchthat cytochrome c oxidation state, by itself or in concert with otherparameters of mitochondrial function including but not limited tomitochondrial oxygen consumption, may be an indicator of reactive freeradical damage to mitochondrial components. Accordingly, the inventionprovides various assays designed to test the inhibition of suchoxidative damage by mitochondria protecting agents. The various formssuch assays may take will be appreciated by those familiar with the artand is not intended to be limited by the disclosures herein, includingin the Examples.

[0091] For example by way of illustration and not limitation, Complex IVactivity may be determined using commercially available cytochrome cthat is fully reduced via exposure to excess ascorbate. Cytochrome coxidation may then be monitored spectrophotometrically at 540 nm using astirred cuvette in which the ambient oxygen above the buffer is replacedwith argon Oxygen reduction in the cuvette may be concurrently monitoredusing a micro oxygen electrode with which those skilled in the art willbe familiar. where such an electrode may be inserted into the cuvette ina manner that preserves the argon atmosphere of the sample, for examplethrough a sealed rubber stopper. The reaction may be initiated byaddition of a cell homogenate or, preferably a preparation of isolatedmitochondria, via injection through the rubber stopper. This assay, orothers based on similar principles, may permit correlation ofmitochondrial respiratory activity with structural features of one ormore mitochondrial components. In the assay described here, for example,a defect in complex IV activity may be correlated with an enzymerecognition site.

[0092] The following examples are offered by way of illustration, andnot by way of limitation.

EXAMPLES Example 1 Synthesis and Characterization of RepresentativeAgents

[0093] This example illustrates the synthesis and characterization ofrepresentative agents of this invention.

[0094] A. Synthesis of Aralkylaminoguanidines

[0095] 1. Compound (1)

[0096] To 122 mg (0.5 mmole) of 3,5-di-tert-butyl-4-hydroxybenzaldehydehemihydrate in 4 ml of acetic acid at room temperature was addedaminoguanidine hydrochloride (110.6 mg, 1.0 mmol) and sodiumcyanoborohydride (314 mg, 5 mmole) and the mixture was stirredovernight. The reaction mixture was then added to 50 ml of saturatedsodium bicarbonate, and extracted with ethyl acetate (2×50 ml). Theorganic layer was dried over anhysrous sodium sulfate, and concentrated.The resulting solid was chromatographed over silica gel usingchloroform/methanol/ acetic acid (84:15:1) as eluting solvent to afford94.8 mg of the product as the acetate salt in 54% yield. ¹H NMR (500MHz, CD₃OD): δ 7.14 (s, 2H), 3.83 (s, 2H), 1.97 (s, CH ₃COO⁻), 1.42 (s,18H)

[0097] 2. Compound (4)

[0098] To 122 mg (0.5 mmole) of 3,5-di-tert-butyl-4-hydroxybenzaldehydehemihydrate in 4 ml of acetic acid at room temperature was addedaminoguanidine hydrochloride (110.6 mg, 1.0 mmol), 150 mg ofparaformaldehyde and sodium cyanoborohydride (314 mg, 5 mmole) and themixture was stirred overnight. The reaction mixture was then added to 50ml of saturated sodium bicarbonate, and extracted with ethyl acetate(2×50 ml). The organic layer was dried over anhysrous sodium sulfate,and concentrated. The resulting solid was chromatographed over silicagel using chloroform/methanol/acetic acid (84:15:1) as eluting solventto afford 119 mg of the product as the acetate salt in 65% yield. ¹H NMR(500 MHz, CD₃OD): δ 7.12 (s, 2H), 3.87 (d, 1H), 3.59 (D, 1H), 2.68 (s,3H), 1.96 (s, CHH ₃COO⁻), 1.42 (s, 18H)

[0099] B. Representative Synthesis of Guanidine Compounds from PrimaryAmines

[0100] 1. Compound (2)

[0101] To tyramine (137 mg, 1 mmole) in 1 ml of DMF was added1-H-pyrazole-carboximidine hydrochloride (146 mg, 1 mmole) anddiisopropylethyl amine (DIEA) (174 μl, 1 mmole), and the reactionmixture was stirred at 23_ for 16 hrs. The solvent was removed in vacuounder 40_ C. The resulting crude material was chromatographed oversilica gel using chloroform/methanol/ acetic acid (84:15:1) as elutingsolvent to furnish 159 mg (67%) of the desired product as the acetatesalt. ¹H NMR (500 MHz, CD₃OD): δ 7.07 (d, 2H, J=8.4 Hz), 6.74 (d, 2H,J=8.4 Hz), 3.39 (t, 2H, J=7 Hz), 2.78 (t, 2H, J=7 Hz), 1.97 (s, CH₃COO⁻).

[0102] 2. Compound (5)

[0103]¹H NMR (500 MHz, CD₃OD): δ 6.89 (d, 1H, J=8.1 Hz), 6.87 (d, 1H,1.9 Hz), (dd, 1H, J=8.1, 1.9 Hz), 3.83 (s, 3H, OCH ₃), 3.80 (s, 3H, OCH₃), 3.43 (t, 2H, J=7.1 Hz), 2.82 (t, 2H, J=7.1 Hz), 1.97 (s, CH ₃COO⁻).

[0104] C. Reaction Scheme for Preparing Arylguanidine Derivatives.

[0105] The following compounds were made according to the followingreaction scheme:

[0106] 1. Compound (7)

[0107] To a round-bottomed flask fitted with an argon inlet were placed1-amino-4-bromonaphthalene (222 mg, 1.0 mmole),1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea (305 mg, 1.05mmole) and dry N,N-dimethylformamide (5 ml). To the above stirredsolution at room temperature were added triethylamine (0.42 ml, 3.0mmole) and mercuric chloride (298 mg, 1.1 mmole). The resulting mixturewas stirred at room temperature, whereupon a white precipitate soonformed. After stirring for 3 h, the reaction mixture was diluted withethyl acetate and filtered through a pad of Celite. The filtrate waswashed with 5% aqueous sodium carbonate (×20 ml), water (2×20 ml) andbrine (1×20 ml). The solution was dried over anhydrous magnesium sulfateand concentrated to provide the crude product. Purification by flashchromatography using 12% ethyl acetate/hexane provided 289 mg of theBoc-protected guanidine derivative in 62% yield. ¹H NMR (500 MHz,CD₃OD): 6 8.27 (m, 1H), 8.02 (m, 2H), 7.84 (d, 1H, J=8.1 Hz), 7.71 84(d, 1H, J=8.1 Hz), 7.67 (m, 2H), 1.61 (s, 9H), 1.35 (s, 9H).

[0108] Deprotection of the Boc group was achieved by treatment withtrifluoroacetic acid (TFA). Thus, to 51 mg (0.11 mmole) of the naphthylderivative under argon was added 1 ml of 50% TFA/CH₂CL₂ solution and themixture was stirred for 3 h at 23° C. The solvent was then removed byrotary evaporation. The crude product was purified by flashchromatography using chloroform/methanol/acetic acid (81:18: 1) aseluting solvent to provided 30 mg of the acetate salt of4-bromo-1-guanidino-naphthalene in 85% yield. ¹H NMR (500 MHz, CD₃OD): δ7.93 (dd, 1H, J=6.6, 2.1 Hz), 8.01 (dd, 1H, J=6.6, 2.1 Hz), 7.92 (d, 1H,J=8 Hz), 7.74 (m, 2H), 7.42 (d, 1H, J=7.7 Hz), 1.95 (s, CH ₃COO⁻).

[0109] 2. Compound (IO)

[0110]¹H NMR (500 MHz, CD₃OD): 6 7.42 (m, 2H), 7.37, (m, 2H), 7.30 (m,1H), 4.83 (m, 1H), 3.43 (dd, 1H, J=3.7, 13.9 Hz), 3.35 (dd, 1H, J=7.5,13.9 Hz).

[0111] 3. Compound (6)

[0112]¹H NMR (500 MHz, CD₃OD): δ 7.04 (dd, 1H, J=2.1, 8.3 Hz), 6.98 (d,1H, J=2.1 Hz), 6.87 (d, 1H, J=8.3 Hz), 2.54 (t, 2H, 7.6 Hz), 1.58 (br.t, 2H), 1.30 (m, 10 H), 0.89 (t, 3H, J=7 Hz).

Example 2 DCFC Assay for Inhibition of ROS Production by MitochondriaProtecting Agents

[0113] In the cell-based aspect of the DCFC assay, monolayers ofcultured adherent SH-SY5Y human neuroblastoma cells (Biedler et al.,Cancer Res. 33:2643, 1973) at or near confluence are rinsed andharvested using trypsin according to standard methods. Single cellsuspensions containing 7.5×10⁴ cells in 200 μl of medium are seeded into96-well plates for overnight incubation at 37° C. and 5% CO₂ in ahumidified cell atmosphere. The following day the wells are gentlyrinsed once with warm Hanks balanced saline solution (HBSS, Gibco-BRL),200 μl of 30 μM dichlorofluorescin-diacetate (DCFC-DA, Molecular Probes,Eugene, Oreg.) are added to each well and cultures are incubated for 2hours at 37° Cl 5% CO₂. The excess DCFC-DA is removed by needleaspiration and each well is gently rinsed twice with HBSS. Each wellthen receives 80 μl of HBSS and 10 μl of mitochondria protecting agent,or vehicle control, diluted into HBSS from stock solutions ofdimethylformamide or dimethylsulfoxide. The final concentration of theorganic solvent is maintained at or below 0.1% (v/v) in HBSS while incontact with cells.

[0114] Cells are equilibrated for 15 minutes at room temperature withthe mitochondria protecting agent (or vehicle control) and then 10 μl offresh 500 μM ferric chloride/300 μM ascorbate solution is added toinitiate free radical formation. Fluorescence of each microculture inthe 96-well plate is quantified immediately using aCytofluorfluorimetric plate reader (model #2350, Millipore Corp.,Bedford, Mass.; excitation wavelength=485 nm; emission wavelength=530nm) and to fluorescence is recorded. The 96-well plates are incubated 30minutes at 37° C./5% CO₂ and fluorescence at 530 nm is again measured(t₃₀). The change in relative mean fluorescence (RMF) over the 30 minuteperiod is calculated for each well.

[0115] The cells are then harvested by trypsinization and counted usinga hemacytometer in order to normalize the data as A(t₃₀−t₀)RMF per cell.The efficacy of a candidate mitochondria protecting agent is determinedby comparing its ability to inhibit ROS production relative to thevehicle control.

[0116] In the cell-free aspect of the DCFC assay, candidate mitochondriaprotecting agents are further evaluated for their ability to inhibit ROSoxidation of DCFC in solution in a microtitre plate format. Stockcompound solutions are usually prepared in dimethylformamide (DMF) ordimethylsulfoxide (DMSO) and diluted further into working concentrationsusing HBSS. Inhibition studies are carried out over a range ofconcentrations. Ten μl of the compound solution or vehicle control and10 μl of a 300 μM DCFC solution in HBSS buffer are added to 60 μl ofHBSS buffer. Ten μl of fresh 500 μM ferric chloride/300 μM ascorbatesolution is then added to initiate free radical formation. Fluorescenceof each well in the 96-well plate is quantified immediately using aCytofluor fluorimetric plate reader (model #2350, Millipore Corp.,Bedford, Mass.; excitation wavelength=485 rim; emission wavelength=530nm) and to fluorescence is recorded. Ten μl of a 0.5% aqueous H₂O₂solution is then added to initiate hydroxyl radical formation throughFenton chemistry and a second fluorimetric reading is taken after 10min. The concentration at which a candidate mitochondria protectingagent exerts 50% of its maximal inhibitory activity (IC₅₀) is calculatedfrom a two-dimensional plot of relative fluorescence units againstinhibitor concentration.

Example 3 Assay for Mitochondrial Permeability Transition Using DASPMI

[0117] The fluorescent mitochondria-selective dye2-,4-dimethylaminostyryl-N-methylpyridinium (DASPMI, Molecular Probes,Inc., Eugene, Oreg.) is dissolved in HBSS at 1 mM and diluted to 25 μMin warm HBSS. In 96-well microculture plates, cultured human cytoplasmichybrid (“cybrid”) cells produced by fusing mitochondrial DNA depleted(ρ⁰) SY5Y cells and mitochondria source platelets (Miller et al., J.Neurochem. 67:1897-1907, 1996) from an individual known or suspected ofhaving a mitochondria associated disease, or from normal (control)platelets, are incubated for 0.5-1.5 hrs in 25 μM DASPMI in a humidified37 C/5% CO₂ incubator to permit mitochondrial uptake of the fluorescentdye. Culture supernatants are then removed and various concentrations ofcandidate mitochondria protecting agents diluted into HBSS from DMFstocks, or vehicle controls, are added at various concentrations.Mitochondria protecting agents are introduced to cells either before, orat the same time as, introduction of the cells to ionomycin (describedbelow).

[0118] Fluorescence of each microculture in the 96-well plate isquantified immediately using a Molecular Devices fmax™ fluorimetricplate reader (Molecular Devices Corp., Sunnyvale, Calif.; excitationwavelength=485 nm; emission wavelength=590 nm) and to fluorescence isrecorded. Thereafter, induction of mitochondrial membrane potentialcollapse is initiated by the addition of ionomycin (Calbiochem, SanDiego, Calif.). Ionomycin stock solutions of various concentrations from0.1-40 μM are prepared in warm Hank's balanced salt solution (HBSS) anddiluted for addition to cells to achieve a final concentration of0.05-20 μM, with final concentrations of 4-10 μM being preferred.Fluorescence decay of DASPMI-loaded, ionomycin induced cells ismonitored as a function of time from 0-500 seconds following addition ofionomycin. The maximum negative slope (V-max) is calculated from asubset of the data using analysis software provided by the fluorimetricplate reader manufacturer. In addition, the initial and final signalintensities are determined and the effects of candidate mitochondriaprotecting agents on the rate of signal decay are quantified.

[0119] Representative data providing IC₅₀ values of mitochondriaprotecting agents are presented below in Table 3. TABLE 3 IC₅₀ Valuesfor Representative Compounds Compound IC₅₀ Cell (μM) Creatine 2000Cyclocreatine 3000 4-Guanidinobenzoic Acid 1000 (2) 100 (4) 10 (11) 100

Example 4 Effect of Agents on Apoptosis

[0120] In 96-well microculture plates, cultured human cells from anindividual known or suspected of having a mitochondria associateddisease, or normal (control) cells or cell lines, are cultured for asuitable period in the presence or absence of physiological inducers ofapoptosis (e.g., Fas ligand, TNF-α, or other inducers of apoptosis knownin the art) and in the presence or absence of representative compoundsof this invention.

[0121] Exteriorization of plasma membrane phosphatidyl serine (PS) isassessed by adding to the 96 well plate annexin-fluoresceinisothiocyanate conjugate (annexin-FITC, Oncogene Research Products,Cambridge, Mass.) dissolved in a suitable buffer for binding to cellsurfaces at a final concentration of 5 μg/well. (Martin et al., J. Exp.Med. 182:1545, 1995) After 15-30 min in a humidified 37° C./5% CO₂incubator, cells are fixed in situ using 2% formalin, washed to removenon-specifically bound FITC and read using a Cytofluor fluorimetricplate reader (model #2350, Millipore Corp., Bedford, Mass.; excitationwavelength=485 nm; emission wavelength=530 nm) to quantify cell surfacebound annexin-FITC as a measure of outer leaflet PS, a marker for cellsundergoing apoptosis.

[0122] Caspase-3 activity is assessed by diluting the fluorogenicpeptide substrate Asp-Glu-Val-Asp-AMC (DEVD-AMC) from a DMSO stocksolution into culture media to a final concentration of 20 μM for uptakeby cells. Substrate cleavage liberates the fluorophore, which ismeasured continuously using a Cytofluor fluorimetric plate reader (model#2350, Millipore Corp., Bedford, Mass.; excitation wavelength=4355 nm;emission wavelength=460 nm). Caspase-1 is measured using the sameprotocol as that for caspase-3, except the caspase-1 specificfluorogenic substrate Tyr-Val-Ala-Asp-Z (Z-YVAD), is substituted forDEVD-AMC and fluorimetry is conducted using 405 nm excitation and 510 nmemission.

[0123] Cytochrome c released from mitochondria of cells undergoingapoptosis is recovered from the post-mitochondrial supernatant andquantified by reverse phase HPLC using a C-18 column, gradient elution(0-45% methanol in phosphate buffer, pH 7.4) and UV absorbance at 254nm. Commercially-obtained authentic cytochrome c serves as the standard.Recovered cytochrome c is also quantified immunochemically by immunoblotanalysis of electrophoretically separated post-mitochondrial supernatantproteins from apoptotic cells, using cytochrome c-specific antibodiesaccording to standard and well accepted methodologies.

Example 5 Effect of Representative Compound on Ionomycin-InducedApoptosis in Neuroblastoma Cells

[0124] SH-SY5Y neuroblastoma cells (1×10⁵ cells) were rinsed with onevolume 1X PBS, and then treated with 10 μM ionomycin (Calbiochem, SanDiego, Cass.) in DMEM supplemented with 10% fetal calf serum (FCS)(Gibco, Life Technologies, Grand Island, N.Y.) for 10 minutes, followedby two washes with DMEM (10% FCS). After 6h incubation at 37° C. in DMEM(10% FCS), cells were visualized by light microscopy (20Xmagnification). Approximately 80% of ionomycin treated cells exhibitedmembrane blebbing, indicative of entry by those cells into a final stageof apoptosis, compared to negligible apoptosis (<5%) in untreated cells.When cells were simultaneously treated with ionomycin and 2 mM creatine,the proportion of cells undergoing apoptosis as evidenced by membraneblebbing was reduced to approximately 10%. Compound (11) at 100 μMprovides the same magnitude of protection from induction of apoptosis asdid 2 mM creatine in this ionomycin induced apoptosis assay.

Example 6 Effect of Representative Compound on Ionomycin InducedApoptosis in Cybrid Cells

[0125] Control cybrid cells (MixCon) produced by fusing ρ⁰ SH-SY5Yneuroblastoma cells with mitochondria source platelets from normalsubjects, and 1685 cells, a cybrid cell line produced by fusing ρ⁰SH-SY5Y cells with mitochondria source platelets from an Alzheimer'sDisease patient (Miller et al., J. Neurochem. 67:1897-1907, 1996), weregrown to complete confluency in 6-well plates (˜3×10⁶ cells/well). Cellswere first rinsed with one volume 1X PBS, and then treated with 10 μMionomycin in the absence or presence of 100 μM compound (12), in DMEMsupplemented with 10% FCS, for 1 minute. At one minute, cells wererinsed twice with five volumes of cold 1X PBS containing a cocktail ofprotease inhibitors (2 μg/ml pepstatin, leupeptin, aprotinin, and 0.1 mMPMSF). Cells were then collected in one ml of cold cytosolic extractionbuffer (210 mM mannitol, 70 mM mannitol, 5 mM each of HEPES, EGTA,glutamate and malate, 1 mM MgCl₂, and the protease inhibitor cocktail atthe concentrations given above. Homogenization was carried out using atype B dounce homogenizer, 25X on ice. Cells were spun at high speed inan Eppendorf microfuge for five minutes to separate cytosol from intactcells, as well as cell membranes and organelles. The supernatant wascollected and an aliquot was saved, along with the pellet, at −80° C.for citrate synthase and protein assays.

[0126] Cytochrome c antibody was covalently bound to solid support chipscontaining a pre-activated surface (ProteinChip, Ciphergen, Palo Alto,Calif.). The spot to be treated with antibody was initially hydratedwith 1 μl of 50% CH₃CN and the antibody solution was added before theCH₃CN evaporated. The concentration of the antibody was approximately 1mg/ml in either Na₃PO₄ or PBS buffer (pH 8.0). The chip was placed in ahumid chamber and stored at 4° C. overnight. Prior to addition of thecytosolic extract, residual active sites were blocked by treatment with1.5 M ethanolamine (pH 8.0) for thirty minutes. The ethanolaminesolution was removed and the entire chip was washed in a 15 ml conicaltube with 10 ml 0.05% Triton-X 100 in 1X PBS, for 5 minutes with gentleshaking at room temperature. The wash buffer was removed and the chipwas sequentially washed, first with 10 ml 0.5 M NaCl in 0.1 M NaOAc (pH4.5), and then with 0.5 M NaCl in 0.1 M Tris (pH 8.0). After removal ofthe Tris-saline buffer, the chip was rinsed with 1× PBS and was readyfor capture of the antigen.

[0127] Fresh supernatant samples were spotted onto the CiphergenProteinChip containing covalently-linked anti-cytochrome c antibody(Pharmingen, San Diego, Calif.). For optimal antibody-cytochrome cinteraction, 100 μl of the supernatant was used and the incubation wascarried out overnight with shaking at 4° C. in a Ciphergen bioprocessingunit. The supernatant was then removed and the spots on the chip werewashed in the bioprocessing unit three times with 200 μl of 0.1%Triton-X 100 in 1X PBS, and then twice with 200 μl of 3.0 M urea in 1XPBS. The chips were then removed from the bioprocessor and washed withapproximately 10 ml of dH₂O. The chips were then dried at roomtemperature prior to the addition of EAM solution (e.g., sinapinic acid,Ciphergen, Palo Alto, Calif.). A suspension of the EAM was made at aconcentration of 25 mg/ml in 50% CH₃CN/H₂O containing 0.5% TFA. Thesaturated EAM solution was clarified by centrifugation and thesupernatant was used for spotting on the ProteinChip surface. Prior tothe addition of EAM to the chip, an internal standard of ubiqutin wasadded to the EAM solution to provide a final concentration of 1 pmol/μl.The quantification of cytochrome c released from mitochondria uponionomycin treatment was based on normalization to the ubiquitin peak inthe mass spectrum and the protein content of the cytosolic extracts.Citrate synthase activity of cytosolic extracts was measured to rule outthe possibility of mitochondrial lysis during the sample preparationprocedure.

[0128] Representative data depicting cytochrome c release in cellsundergoing ionomycin induced apoptosis, and attenuation of cytochromerelease in cells treated with 100 μM compound (1 1) at the same timeionomycin was introduced, are presented in the FIGURE.

Example 7 Effect of Representative Compounds on Thapsigargin InducedApoptosis

[0129] In order to determine the effect of compounds of this inventionon the final endpoint of apoptosis (cell death), the following assayswere carried out. The cells used were 1685 cells, “1685” being thedesignation of a cybrid cell line derived from SH-SY5Y and containingmitochondria from a patient having Alzheimer's disease (see U.S. Pat.No. 5,888,498, issued Mar. 30, 1999, hereby incorporated by reference).Cells were plated (3×10⁴ cells per well) on 96-well plates 48 hoursprior to thapsigargin treatment. Thapsigargin (Calbiochem, La Jolla,Calif.) alone (final concentration, 1 μM), thapsigargin (1 μM) plusagent final concentration, 100 μM), agent alone (100 μM) in growthmedia, or growth media devoid of both thapsigargin and agent, were addedto cells in four separate wells.

[0130] Twenty-four hours after thapsigargin +/− agent treatment,propidium iodide (Sigma Chemical Co., St. Louis, Mo.) was added to eachwell at a final concentration of 10 μg/ml per well. The cells wereincubated at ambient temperature for 10 minutes, after which thefluorescence (excitation max=536 nm, 544 nm used for excitation;emission max=617, readings at 612 nm) was determined for each individualwell in a fmax™ fluorescence microplate reader (Molecular Devices,Sunnyvale, Calif.). The resulting fluorescence values correspond tocells in the monolayer of a well that are non-viable.

[0131] Next, the media was aspirated, and the monolayer was fixed(killed) by adding 100 μl of 100% ethanol to each well followed byincubation at ambient temperature for 10 minutes. The fluorescense ineach well was then read again. The fluorescence values resulting fromthe second reading correspond to the total number of cells (whetherviable or non-viable at the time of the initial fluorescence reading)present in the monolayer of a well.

[0132] The results, presented in Table 4 below, are expressed as thepercentage of non-viable cells as a proportion of the viable cells(fixed controls). TABLE 4 Effect of Representative Compounds onThapsigargin-Induced Apoptosis % Non-Viable % Non-Viable Cells withCells with Cpd. Thapsigargin and Thapsigargin and Δ Cell No. withoutCmpd. with Cmpd. Viability¹ P-Value²  (1) 41.5 38.3 3.2 0.0978  (3) 41.746.7 −5.0 0.2120  (4) 45.9 43.8 2.1 0.5908  (6) 44.0 122.1 −78.1 <0.0001 (9) 49.6 42.0 7.6 0.1794 (11) 36.0 29.0 7.0 0.0977 (12) 39.3 37.5 1.80.3268 (13) 37.0 52.8 −15.8 <0.0001 (14) 44.4 47.9 −3.5 0.0681 (15) 39.142.2 −3.1 0.1132 (16) 46.6 121.2 −74.6 <0.0001 (17) 45.2 56.9 −11.7<0.0001 (18) 46.5 47.8 −1.3 0.5853 (19) 41.9 44.3 −2.4 0.3963 (20) 45.448.9 −3.5 0.2714 (21) 44.1 43.8 0.3 0.9418 (22) 47.8 53.8 −6.0 0.0015(23) 50.4 49.8 0.6 0.7626 (24) 47.8 47.1 0.7 0.7476 (25) 52.0 48.9 3.10.3249 (26) 50.7 38.0 12.7 <0.0001 (27) 50.4 42.5 7.9 0.0273 (28) 48.852.3 −3.5 0.0950 (29) 45.7 48.6 −2.9 0.0908 (30) 47.3 36.5 10.8 <0.0001(31) 45.3 100.0 −54.7 <0.0001 (32) 45.8 45.7 0.1 0.9352 (33) 50.6 49.21.4 0.6732 (34) 45.3 42.4 2.9 0.1743 (35) 45.1 42.3 2.8 0.0865

[0133] The data presented in Table 4, and other results from theseexperiments, define classes of compounds, i.e., (1) anti-apoptotic orthapsigargin protective agents;

[0134] (2) pro-apoptotic or thapsigargin enhancing agents; (3) cytotoxicagents; and (4) agents that have little or no impact on the apoptoticeffects of thapsigargin. Each of these classes of compounds is describedin more detail infra.

[0135] Class 1: Anti-apoptotic or thapsigargin protective agents.

[0136] These agents have a ΔCell Viability that is a positive number;this indicates that a lower percentage of cells undergo apoptosis due tothapsigargin treatment when the agent is present than when it is not.Agents in Class 1 include Compounds (11), (9), (30), (27), and (26).These compounds have a ΔCell Viability value≧about +4.5.

[0137] Class 2: Pro-apoptotic or thapsigargin enhancing agents. Theseagents have a ΔCell Viability that is a negative number, which indicatesthat a higher percentage of cells undergo apoptosis due to thapsigargintreatment when the agent is present than when it is not. Agents in Class2 include Compounds (22) and (3). These compounds have a ΔCell Viabilityvalue≦−4.5 and ≧about −10.

[0138] Class 3: Cytotoxic Agents.

[0139] These agents have a ΔCell Viability in the presence ofthapsigargin that is a large negative number, which might indicate thatthese agents are strongly pro- apoptotic or thapsigargin enhancingagents, i.e., that a much higher percentage of cells undergo apoptosisdue to thapsigargin treatment when the agent is present than when it isnot. However, because these agents significantly increase the percentageof non-viable cells even in the absence of thapsigargin (see Table 4below), they are designated cytotoxic agents. Agents in Class 3 includeCompounds (6), (31), (17), (16) and (13). These compounds have a ΔCellViability [(+thapsigargin, −compound)—(+thapsigargin, +compound)] value≦ about −10, ranging from about −12 to −-16 (Compounds (17) and (13))down to about −55 (Compound (31)) and about −75 to −80 (Compounds (6)and (16)) and lower.

[0140] The cytotoxic nature of these compounds is revealed by the ΔCellViability values resulting from treatment of cells with the compound inthe absence of thapsigargin, as detailed in Table 5. The ΔCell Viabilityvalues resulting from treating cells with agents in this class in theabsence of thapsigargin (Table 4, infra) closely parallel the ΔCellViability values that result when cells are treated with the respectiveagent and thapsigargin (Table 4, supra). This indicates that compoundsin this class exert their effect predominately by being cytotoxic, andthat they may have little or no thapsigargin enhancing activity. Incontrast, agents in Classes 1, 2 and 4 do not exhibit these cytotoxiceffects. TABLE 5 Cytotoxic Effects of Representative Compounds (NoThapsigargin) Cpd. % Non-Viable Cells % Non-Viable Cells Δ Cell No.without Cmpd. with Cmpd. Viability P-Value²  (6) 16.2 125.2 −109.0<0.0001 (13) 17.2 39.4 −22.2 <0.0001 (16) 17.0 123.7 −106.7 <0.0001 (17)17.7 41.3 −23.6 <0.0001 (31) 16.5 100.0 −83.5 <0.0001

[0141] Class 4: Agents Having Little or No Effect on ThapsigarginInduced Apoptosis.

[0142] These agents have ΔCell Viability values that relatively smallpositive or negative numbers, which indicates that the percentage ofcells undergoing apoptosis due to thapsigargin treatment when the agentis present is not much different than when it is not. Compounds in Class4 have a ΔCell Viability value ranging from about 3.5 (Compound (1)) toabout −3.5 (Compounds (20) and (14)).

[0143] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein forpurposes of illustration, various' modifications may be made withoutdeviating from the spirit and scope of the invention. Accordingly, theinvention is not limited except as by the appended claims.

1. A method for treating a mitochondria-associated disease byadministering to a warm-blooded animal in need thereof an effectiveamount of a compound having the following structure:

including stereoisomers, prodrugs and pharmaceutically acceptable saltsthereof, wherein: Ar is phenyl or naphthyl optionally substituted with 1to 5 R₂ groups; L is an optional linker moiety selected from—(CH₂)_(n)—, —(CH₂)_(n)NH—, —(CH₂)_(n)N(C₁₋₄alkyl)—, —NHC(═NH)— and—(CH₂)_(n)O(CH₂)_(n)—, wherein n is 1-4 and each linker moiety isoptionally substituted with 1 to 5 R₃ groups; R₂ is hydroxy, C₁₋₁₂alkyl,C₁₋₁₂alkyloxy, halo, —NH₂, —NHR, —NRR, cyano, nitro, —SR, —COOH,C₇₋₁₂aralkyl or heterocycle; or C₁₋₂alkyl, C₁₋₁₂alkyloxy, —NH₂, —NHR,—NRR, —SR, C₇₋₁₂aralkyl or heterocycle substituted with 1 to 5 R₃groups; R₃ is hydroxy, halo, C₁₋₄alkyl, —OR, —NH₂, —NHR or —NRR; andeach occurrence of R is independently selected from C₁₋₄alkyl.
 2. Themethod of claim 1 wherein Ar is phenyl optionally substituted with 1 to5 R₂ groups.
 3. The method of claim 2 wherein Ar is phenyl,3,5-di-t-butyl-4-hydroxyphenyl. 2-methoxy-4-carboxylphenyl,2-chloro-4-carboxyl-5-methoxyphenyl, 3,5-di-tetrafluoromethylphenyl,3,5-difluorophenyl, 3,4,5-trimethoxyphenyl, 4-n-hexoxyphenyl,4-fluorophenyl, 3-trifluorophenyl, 2-carbinolphenyl,2-chloro-5-methylphenyl, 3-carboxylphenyl, 3-carboxyl-4-hydroxyphenyl,2-methyl-4-carboxylphenyl, 4-methoxyphenyl, 2-hydroxyphenyl,4-(N-morphinol)phenyl, 3,4-dihydroxyphenyl, 2,4-dimethylphenyl,2-methyl-4-hydroxyphenyl, 4-n-octylphenyl, 2-hydroxy-5-n-octylphenyl,4-chlorophenyl, or 2-methyl-4-chlorophenyl,
 4. The method of claim 1wherein Ar is naphthyl optionally substituted with 1 to 5 R₂ groups. 5.The method of claim 4 wherein Ar is naphthyl or 4-bromonaphthyl.
 6. Themethod of claim 1 wherein the L is not present.
 7. The method of claim 1wherein L is present.
 8. The method of claim 7 wherein L is —CH₂NH—,—CH₂CH₂, —CH(OH)CH₂—, —CH₂N(CH₃)— or —NHC(═NH)—.
 9. The method of claim1 wherein the compound is administered in the form of a pharmaceuticalcomposition.
 10. The method of claim 1 wherein themitochondria-associated disease is a disease in which free radicalmediated oxidative injury leads to tissue degeneration.
 11. The methodof claim 1 wherein the mitochondria-associated disease is a disease inwhich cells inappropriately undergo apoptosis.
 12. The method of claim10 or 11 wherein the mitochondria-associated disease is Alzheimer'sDisease, Parkinson's Disease, Huntington's Disease, auto-immune disease,diabetes mellitus (Type I or Type II), congenital muscular dystrophy,fatal infantile myopathy, “later-onset” myopathy, MELAS (mitochondrialencephalopathy, lactic acidosis, and stroke), MIDD (mitochondrialdiabetes and deafness), MERFF (myoclonic epilepsy ragged red fibersyndrome), arthritis, NARP (Neuropathy; Ataxia; Retinitis Pigmentosa),MNGIE (Myopathy and external ophthalmoplegia; Neuropathy;Gastro-Intestinal; Encephalopathy), LHON (Leber's; Hereditary; Optic;Neuropathy), Kearns-Sayre disease, Pearson's Syndrome, PEO (ProgressiveExternal Ophthalmoplegia), Wolfram syndrome, DIDMOAD (DiabetesInsipidus, Diabetes Mellitus, Optic Atrophy, Deafness), Leigh'sSyndrome, dystonia, or schizophrenia.
 13. The method of claim 1 whereinthe mitochondria-associated disease is a disease in which cells fail toundergo apoptosis.
 14. The method of claim 13 wherein themitochondria-associated disease is cancer.
 15. The method of claim 1wherein the mitochondria-associated disease is stroke.
 16. The method ofclaim 1 wherein the mitochondria-associated disease is Alzheimer'sDisease.
 17. The method of claim 1 wherein the mitochondria-associateddisease is diabetes.
 18. The method of claim 1 wherein themitochondria-associated disease is auto-immune disease.
 19. The methodof claim 1 wherein the mitochondria-associated disease is psoriasis. 20.A pharmaceutical composition comprising a compound having the followingstructure:

including stereoisomers, prodrugs and pharmaceutically acceptable saltsthereof, wherein: Ar is phenyl or naphthyl optionally substituted with 1to 5 R₂ groups; L is an optional linker moiety selected from—(CH₂)_(n)—, —(CH₂)_(n)NH—, —(CH₂)_(n)N(C₁₋₄alkyl)—, —NHC(═NH)— and—(CH₂)_(n)O(CH₂)_(n)—, wherein n is 1-4 and each linker moiety isoptionally substituted with 1 to 5 R₃ groups; R₂ is hydroxy, C₁₋₁₂alkyl,C₁₋₁₂alkyloxy, halo, —NH₂, —NHR, —NRR, cyano, nitro, —SR, —COOH,C₇₋₁₂aralkyl or heterocycle; or C₁₋₁₂alkyl, C₁₋₁₂alkyloxy, —NH₂, —NHR,—NRR, —SR, C₇₋₁₂aralkyl or heterocycle substituted with 1 to 5 R₃groups; R₃ is hydroxy, halo, C₁₋₄alkyl, —OR, —NH₂, —NHR or —NRR; andeach occurrence of R is independently selected from C₁₋₄alkyl; and apharmaceutically acceptable carrier.